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A proposal has been posted on the ICTV website (2011.001aG.N.v1.binomial_sp_names) to replace virus species names by non-Latinized binomial names consisting of the current italicized species name with the terminal word "virus" replaced by the italicized and non-capitalized genus name to which the species belongs. If implemented, the current italicized species name Measles virus, for instance, would become Measles morbillivirus while the current virus name measles virus and its abbreviation MeV would remain unchanged. The rationale for the proposed change is presented.

The ageing population of pet dogs and cats constitutes a group of animals that requires specific veterinary attention; particularly with respect to the prevention of infectious diseases. Although geriatric medicine for these species has received some recent attention; there has been little study of the vaccination requirements of such animals. A continuing challenge to the veterinary profession is the application of the principles of evidence-based medicine to the immune protection of companion animals. Recently, national and international vaccination guidelines have been developed to provide advice to the veterinary practitioner. There has been some reluctance to embrace these recommendations and an opinionated clientele has continued to delay acceptance of these recent insights. As has been the case in human medicine, antagonism and pessimism towards vaccination also occur in veterinary vaccinology, both for the young and the ageing animal., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Overview: Feline calicivirus (FCV) is a highly variable virus. More severe, systemic forms of FCV infection have been observed recently., Infection: Sick, acutely infected or carrier cats shed FCV in oronasal and conjunctival secretions. Infection occurs mainly through direct contact., Disease Signs: The main clinical signs are oral ulcers, upper respiratory signs and a high fever. Feline calicivirus may be isolated from nearly all cats with chronic stomatitis or gingivitis. Cats with 'virulent systemic FCV disease' variably show pyrexia, cutaneous oedema, ulcerative lesions on the head and limbs, and jaundice. Mortality is high and the disease is more severe in adult cats., Diagnosis: Diagnosis of FCV can be achieved by virus isolation or reverse-transcriptase PCR. Viral RNA can be detected in conjunctival and oral swabs, blood, skin scrapings or lung tissue using PCR. Positive PCR results should be interpreted with caution, as these may be a consequence of low-level shedding by persistently infected carriers. The diagnosis of virulent systemic FCV disease relies on clinical signs and isolation of the same strain from the blood of several diseased cats., Disease Management: Supportive therapy (including fluid therapy) and good nursing care are essential. Anorexic cats should be fed highly palatable, blended or warmed food. Mucolytic drugs (eg, bromhexine) or nebulisation with saline may offer relief. Broad-spectrum antibiotics may be administered to prevent secondary bacterial infections. Feline calicivirus can persist in the environment for about 1 month and is resistant to many common disinfectants., Vaccination Recommendations: Two injections, at 9 and 12 weeks of age, are recommended, followed by a first booster 1 year later. In high-risk situations, a third vaccination at 16 weeks is recommended. Boosters should be given every 3 years. However, cats in high-risk situations should be revaccinated annually. Cats that have recovered from caliciviral disease are probably not protected for life, particularly if infected with different strains. Vaccination of these cats is still recommended.

Overview: Feline immunodeficiency virus (FIV) is a retrovirus closely related to human immunodeficiency virus. Most felids are susceptible to FIV, but humans are not. Feline immunodeficiency virus is endemic in domestic cat populations worldwide. The virus loses infectivity quickly outside the host and is susceptible to all disinfectants., Infection: Feline immunodeficiency virus is transmitted via bites. The risk of transmission is low in households with socially well-adapted cats. Transmission from mother to kittens may occur, especially if the queen is undergoing an acute infection. Cats with FIV are persistently infected in spite of their ability to mount antibody and cell-mediated immune responses., Disease Signs: Infected cats generally remain free of clinical signs for several years, and some cats never develop disease, depending on the infecting isolate. Most clinical signs are the consequence of immunodeficiency and secondary infection. Typical manifestations are chronic gingivostomatitis, chronic rhinitis, lymphadenopathy, weight loss and immune-mediated glomerulonephritis., Diagnosis: Positive in-practice ELISA results obtained in a low-prevalence or low-risk population should always be confirmed by a laboratory. Western blot is the 'gold standard' laboratory test for FIV serology. PCR-based assays vary in performance., Disease Management: Cats should never be euthanased solely on the basis of an FIV-positive test result. Cats infected with FIV may live as long as uninfected cats, with appropriate management. Asymptomatic FIV-infected cats should be neutered to avoid fighting and virus transmission. Infected cats should receive regular veterinary health checks. They can be housed in the same ward as other patients, but should be kept in individual cages., Vaccination Recommendations: At present, there is no FIV vaccine commercially available in Europe. Potential benefits and risks of vaccinating FIV-infected cats should be assessed on an individual cat basis. Needles and surgical instruments used on FIV-positive cats may transmit the virus to other cats, so strict hygiene is essential.

Overview: Rabies virus belongs to the genus Lyssavirus, together with European bat lyssaviruses 1 and 2. In clinical practice, rabies virus is easily inactivated by detergent-based disinfectants., Infection: Rabid animals are the only source of infection. Virus is shed in the saliva some days before the onset of clinical signs and transmitted through a bite or a scratch to the skin or mucous membranes. The average incubation period in cats is 2 months, but may vary from 2 weeks to several months, or even years., Disease Signs: Any unexplained aggressive behaviour or sudden behavioural change in cats must be considered suspicious. Two disease manifestations have been identified in cats: the furious and the dumb form. Death occurs after a clinical course of 1-10 days., Diagnosis: A definitive rabies diagnosis is obtained by post-mortem laboratory investigation. However, serological tests are used for post-vaccinal control, especially in the context of international movements., Disease Management: Post-exposure vaccination of cats depends on the national public health regulations, and is forbidden in many countries., Vaccination Recommendations: A single rabies vaccination induces a long-lasting immunity. Kittens should be vaccinated at 12-16 weeks of age to avoid interference from maternally derived antibodies and revaccinated 1 year later. Although some vaccines protect against virulent rabies virus challenge for 3 years or more, national or local legislation may call for annual boosters.

Overview: Avian influenza is a disease of birds, caused by a type A influenza virus. The subtype H5N1 avian influenza occurs primarily in birds and infection varies from mild disease with little or no mortality to a highly fatal, rapidly spreading epidemic (highly pathogenic avian influenza). It is extremely rare for cats to be infected and there are only very few confirmed reports of the disease in cats in Europe., Infection: Cats can be infected via the respiratory and oral routes (eg, by eating infected birds). The key precondition for infection is that the cat lives in an area where H5N1 virus infection has been confirmed in birds. Additionally, the cat should have had outdoor access to an environment where waterfowl is present, or contact with poultry or uncooked poultry meat, or close contact with an H5N1-infected, sick cat during the first week of infection. CLINICAL SUSPICION: Clinical signs in cats may include fever, lethargy, dyspnoea, conjunctivitis and rapid death. Neurological signs (circling, ataxia) have also been recorded., Diagnosis: The veterinary authorities should be notified. Oropharyngeal, nasal and/or rectal swabs or faecal samples of suspected cases should be submitted for PCR and/or virus isolation. Post-mortem samples of lung and mediastinal lymph nodes should be obtained. Particular care should be taken when handling the cat and/or samples., Disease Management: The virus is sensitive to all standard medical disinfectants. Cats with suspected H5N1 infection should be kept in strict isolation. Owners should be advised to confine the cat to a separate room prior to bringing it to the veterinary clinic. VACCINATION AND DISEASE PREVENTION: No H5N1 vaccines are commercially available for cats. In the event of confirmed cases of H5N1 avian influenza in birds in the area, owners should keep their cats indoors until further information is available, and follow official regulations.

Overview: Chlamydophila felis is a Gram-negative bacterium and its primary target is the conjunctiva. The bacterium does not survive outside the host., Infection: Transmission requires close contact between cats; ocular secretions are probably the most important body fluid for infection. Most cases occur in cats under 1 year of age. Chlamydophila felis is the infectious organism most frequently associated with conjunctivitis., Disease Signs: Unilateral ocular disease generally progresses to become bilateral. There can be intense conjunctivitis with extreme hyperaemia of the nictitating membrane, blepharospasm and ocular discomfort. Transient fever, inappetence and weight loss may occur shortly after infection, although most cats remain well and continue to eat., Diagnosis: PCR techniques are now preferred for diagnosing C felis infection. Ocular swabs are generally used. In unvaccinated cats, antibody detection can be used to indicate infection., Disease Management: Tetracyclines are generally regarded as the antibiotics of choice. Doxycycline has the advantage of requiring only single daily administration and is given at a dose of 10 mg/kg orally. Vaccination should be considered if there is a history of confirmed chlamydial disease in a shelter. Single housing and routine hygiene measures should suffice to avoid cross-infection. Cats maintained together for longer terms should be vaccinated regularly. In breeding catteries where C felis infection is endemic, the first step should be to treat all cats with doxycycline for at least 4 weeks. Once clinical signs have been controlled, the cats should be vaccinated., Vaccination Recommendations: Vaccination should be considered for cats at risk of exposure to infection. Vaccination generally begins at 8-10 weeks of age, with a second injection 3-4 weeks later. Annual boosters are recommended for cats at continued risk of exposure.

Overview: Feline viral rhinotracheitis, caused by feline herpesvirus (FHV), is an upper respiratory tract disease that is often associated with feline calicivirus and bacteria. In most cats, FHV remains latent after recovery, and they become lifelong virus carriers. Stress or corticosteroid treatment may lead to virus reactivation and shedding in oronasal and conjunctival secretions., Infection: Sick cats shed FHV in oral, nasal and conjunctival secretions; shedding may last for 3 weeks. Infection requires direct contact with a shedding cat., Disease Signs: Feline herpesvirus infections cause acute rhinitis and conjunctivitis, usually accompanied by fever, depression and anorexia. Affected cats may also develop typical ulcerative, dendritic keratitis., Diagnosis: Samples consist of conjunctival, corneal or oropharyngeal swabs, corneal scrapings or biopsies. It is not recommended that cats recently vaccinated with a modified-live virus vaccine are sampled. Positive PCR results should be interpreted with caution, as they may be produced by low-level shedding or viral latency., Disease Management: 'Tender loving care' from the owner, supportive therapy and good nursing are essential. Anorexic cats should be fed blended, highly palatable food - warmed up if required. Mucolytic drugs (eg, bromhexine) or nebulisation with saline may offer relief. Broad-spectrum antibiotics should be given to prevent secondary bacterial infections. Topical antiviral drugs may be used for the treatment of acute FHV ocular disease. The virus is labile and susceptible to most disinfectants, antiseptics and detergents., Vaccination Recommendations: Two injections, at 9 and 12 weeks of age, are recommended, with a first booster 1 year later. Boosters should be given annually to at-risk cats. For cats in low-risk situations (eg, indoor-only cats), 3-yearly intervals suffice. Cats that have recovered from FHV-associated disease are usually not protected for life against further disease episodes; vaccination of recovered cats is therefore recommended.

Overview: Bordetella bronchiseptica is a Gram-negative bacterium that colonises the respiratory tract of mammals and is considered to be a primary pathogen of domestic cats. It is sensible to consider B bronchiseptica as a rare cause of zoonotic infections. The bacterium is susceptible to common disinfectants., Infection: The bacterium is shed in oral and nasal secretions of infected cats. Dogs with respiratory disease are an infection risk for cats. The microorganism colonises the ciliated epithelium of the respiratory tract of the host, establishing chronic infections., Disease Signs: A wide range of respiratory signs has been associated with B bronchiseptica infection, from a mild illness with fever, coughing, sneezing, ocular discharge and lymphadenopathy to severe pneumonia with dyspnoea, cyanosis and death., Diagnosis: Bacterial culture and PCR lack sensitivity. Samples for isolation can be obtained from the oropharynx (swabs) or via transtracheal wash/ bronchoalveolar lavage., Disease Management: Antibacterial therapy is indicated, even if the signs are mild. Where sensitivity data are unavailable, tetracyclines are recommended. Doxycycline is the antimicrobial of choice. Cats with severe B bronchiseptica infection require supportive therapy and intensive nursing care., Vaccination Recommendations: In some European countries an intranasal modified-live virus vaccine is available. The modified-live product is licensed for use as a single vaccination with annual boosters. Cats should not be routinely vaccinated against B bronchiseptica (non-core), since the infection generally causes only a mild disease.

Overview: Feline leukaemia virus (FeLV) is a retrovirus that may induce depression of the immune system, anaemia and/or lymphoma. Over the past 25 years, the prevalence of FeLV infection has decreased considerably, thanks both to reliable tests for the identification of viraemic carriers and to effective vaccines., Infection: Transmission between cats occurs mainly through friendly contacts, but also through biting. In large groups of non-vaccinated cats, around 30-40% will develop persistent viraemia, 30-40% show transient viraemia and 20-30% seroconvert. Young kittens are especially susceptible to FeLV infection., Disease Signs: The most common signs of persistent FeLV viraemia are immune suppression, anaemia and lymphoma. Less common signs are immune-mediated disease, chronic enteritis, reproductive disorders and peripheral neuropathies. Most persistently viraemic cats die within 2-3 years., Diagnosis: In low-prevalence areas there may be a risk of false-positive results; a doubtful positive test result in a healthy cat should therefore be confirmed, preferably by PCR for provirus. Asymptomatic FeLV-positive cats should be retested., Disease Management: Supportive therapy and good nursing care are required. Secondary infections should be treated promptly. Cats infected with FeLV should remain indoors. Vaccination against common pathogens should be maintained. Inactivated vaccines are recommended. The virus does not survive for long outside the host., Vaccination Recommendations: All cats with an uncertain FeLV status should be tested prior to vaccination. All healthy cats at potential risk of exposure should be vaccinated against FeLV. Kittens should be vaccinated at 8-9 weeks of age, with a second vaccination at 12 weeks, followed by a booster 1 year later. The ABCD suggests that, in cats older than 3-4 years of age, a booster every 2-3 years suffices, in view of the significantly lower susceptibility of older cats.

Overview: Feline coronavirus infection is ubiquitous in domestic cats, and is particularly common where conditions are crowded. While most FCoV-infected cats are healthy or display only a mild enteritis, some go on to develop feline infectious peritonitis, a disease that is especially common in young cats and multi-cat environments. Up to 12% of FCoV-infected cats may succumb to FIP, with stress predisposing to the development of disease., Disease Signs: The 'wet' or effusive form, characterised by polyserositis (abdominal and/or thoracic effusion) and vasculitis, and the 'dry' or non-effusive form (pyogranulomatous lesions in organs) reflect clinical extremes of a continuum. The clinical picture of FIP is highly variable, depending on the distribution of the vasculitis and pyogranulomatous lesions. Fever refractory to antibiotics, lethargy, anorexia and weight loss are common non-specific signs. Ascites is the most obvious manifestation of the effusive form., Diagnosis: The aetiological diagnosis of FIP ante-mortem may be difficult, if not impossible. The background of the cat, its history, the clinical signs, laboratory changes, antibody titres and effusion analysis should all be used to help in decision-making about further diagnostic procedures. At the time of writing, there is no non-invasive confirmatory test available for cats without effusion., Disease Management: In most cases FIP is fatal. Supportive treatment is aimed at suppressing the inflammatory and detrimental immune response. However, there are no controlled studies to prove any beneficial effect of corticosteroids., Vaccination Recommendations: At present, only one (intranasal) FIP vaccine is available, which is considered as being non-core. Kittens may profit from vaccination when they have not been exposed to FCoV (eg, in an early-weaning programme), particularly if they enter a FCoV-endemic environment.

Overview: Feline panleukopenia virus (FPV) infects all felids as well as raccoons, mink and foxes. This pathogen may survive in the environment for several months and is highly resistant to some disinfectants., Infection: Transmission occurs via the faecal-oral route. Indirect contact is the most common route of infection, and FPV may be carried by fomites (shoes, clothing), which means indoor cats are also at risk. Intrauterine virus transmission and infection of neonates can occur., Disease Signs: Cats of all ages may be affected by FPV, but kittens are most susceptible. Mortality rates are high - over 90% in kittens. Signs of disease include diarrhoea, lymphopenia and neutropenia, followed by thrombocytopenia and anaemia, immunosuppression (transient in adult cats), cerebellar ataxia (in kittens only) and abortion., Diagnosis: Feline panleukopenia virus antigen is detected in faeces using commercially available test kits. Specialised laboratories carry out PCR testing on whole blood or faeces. Serological tests are not recommended, as they do not distinguish between infection and vaccination., Disease Management: Supportive therapy and good nursing significantly decrease mortality rates. In cases of enteritis, parenteral administration of a broad-spectrum antibiotic is recommended. Disinfectants containing sodium hypochlorite (bleach), peracetic acid, formaldehyde or sodium hydroxide are effective., Vaccination Recommendations: All cats - including indoor cats - should be vaccinated. Two injections, at 8-9 weeks of age and 3-4 weeks later, are recommended, and a first booster 1 year later. A third vaccination at 16-20 weeks of age is recommended for kittens from environments with a high infection pressure (cat shelters) or from queens with high vaccine-induced antibody levels (breeding catteries). Subsequent booster vaccinations should be administered at intervals of 3 years or more. Modified-live virus vaccines should not be used in pregnant queens or in kittens less than 4 weeks of age.

Background: The European Advisory Board on Cat Diseases (ABCD) is a body of experts that sees its task as bringing feline health issues to the forefront of companion animal practice. By way of an introduction to this special 'clinical practice' issue of the Journal of Feline Medicine and Surgery (JFMS), this article attempts a 'helicopter view' of practical, or applied, immunology. It should be viewed as a 'light primer' to vaccines and vaccination, and is very general in nature. It is not intended to replace authoritive immunology textbooks, which abound both in the veterinary and medical fields, and the level of detail in which may discourage the casual reader. By design, therefore, this article is not referenced., Underpinning Issues: The immune response (be it after vaccination or infection) is discussed, as are the issues of duration of immunity, and vaccine safety and efficacy, tests predicting protection, population ('herd') immunity, and the types of vaccine developed and/or available (live, killed, chimaeric, DNA-only products)., Practical Relevance: With day-to-day veterinary practice in mind, practical issues discussed include kitten vaccination, the definition of 'core' versus 'non-core' products, passive immunisation, and prevention strategies in populations and crowded cat communities. Adverse reactions, and factors affecting vaccine efficacy, safety and performance are also summarised.

Vaccination guidelines are non-compulsory recommendations which assist the veterinary practitioner to use vaccines efficiently. They complement the official information contained in the shortened form of the summary of product characteristics that is included in the package insert of the product. The aim of this article is to clarify the role of guidelines and examine how they can improve the use of vaccines in practical conditions. The development of vaccination guidelines is explained. Several issues are discussed: primary vaccination schedule; interference with maternally derived antibodies; duration of immunity; vaccination and ageing. Three guidelines dealing with the vaccination of cats against upper respiratory tract disease are compared, as an example. In conclusion, vaccination guidelines are essential tools to assist veterinarians in good vaccination practices. They fill the gap that exists between the official recommendations included in the regulations and the licensing dossiers and the daily use of the vaccines., (© World Organisation for Animal Health (OIE), 2007)

The Asian lineage highly pathogenic avian influenza (HPAI) H5N1 virus is a known pathogen of birds. Only recently, the virus has been reported to cause sporadic fatal disease in carnivores, and its zoonotic potential has been dominating the popular media. Attention to felids was drawn by two outbreaks with high mortality in tigers, leopards and other exotic felids in Thailand. Subsequently, domestic cats were found naturally infected and experimentally susceptible to H5N1 virus. A high susceptibility of the dog to H3N8 equine influenza A virus had been reported earlier, and recently also HPAI H5N1 virus has been identified as a canine pathogen. The ferret, hamster and mouse are suitable as experimental animals; importantly, these species are also kept as pets. Experimental intratracheal and oral infection of cats with an HPAI H5N1 virus isolate from a human case resulted in lethal disease; furthermore, cats have been infected by the feeding of infected chickens. Spread of the infection from experimentally infected to in-contact cats has been reported. The epidemiological role of the cat and other pet animal species in transmitting HPAI H5N1 virus to humans needs continuous consideration and attention.

A yearly revaccination of adult pets against distemper, the adenoviral and parvoviral diseases is scientifically unwarranted, professionally obsolete and ethically questionable; other vaccinal antigens, however, may need yearly or even more frequent injections. Base immunisation is redefined: it is complete only after the multivalent booster in the second year of life. A yearly revaccination interview, not necessarily an injection, should become the new standard. This interview is a professional service that must be taught, expertly performed and invoiced. Adult animals should be "vaccinated to measure", taking age, breed, lifestyle, the epidemiologic situation, etc. into account. Post-vaccination serology should become a guide in revaccination decisions. For a solid herd immunity, more animals of the population must be vaccinated. The profession should issue regular updates of the 'code of vaccination practice'. To counteract vaccination antagonism, a concerted action of academia, the veterinary profession and industry is required.

A reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of feline coronavirus (FCoV) messenger RNA in peripheral blood mononuclear cells (PBMCs) is described. The assay is evaluated as a diagnostic test for feline infectious peritonitis (FIP). It is based on a well-documented key event in the development of FIP: the replication of virulent FCoV mutants in monocytes/macrophages. To detect most feline coronavirus field strains, the test was designed to amplify subgenomic mRNA of the highly conserved M gene. The test was applied to 1075 feline blood samples (424 from healthy, 651 from sick cats suspected of FIP) and returned 46% of the diseased cats as positive for feline coronavirus mRNA in their peripheral blood cells; of the healthy cats, 5% tested positive. Of a group of 81 animals in which FIP had been confirmed by post-mortem examination, 75 (93%) tested positive, whereas 17 cats with different pathologies (non-FIP cases) all tested negative. In view of the low rate of false-positive results (high specificity) the mRNA RT-PCR may be a valuable addition to the diagnostic arsenal for FIP.

Dendritic cells (DCs) induce different types of immune responses depending on their lineage and activation signals. When exposed to inactivated pseudorabiesvirus (iPRV), plasmacytoid but not myeloid DCs released IFN-alpha and IL-12. Remarkably, both iPRV-pulsed DC types were able to induce primary IFN-gamma producing T cells and IgG isotype switching in vivo. In contrast, tetanus toxoid pulsed DCs did not induce detectable primary immune responses. The efficacy of antiviral T and B cell priming proved dependent on the recipient's genotype. We conclude that either plasmacytoid or myeloid DCs pulsed with inactivated virus suffice to induce primary Th1-polarised immune responses.

Replicating viruses generally induce type 1 immune responses, with high interferon (IFN)-gamma levels and antibodies of the IgG2a isotype. In the present study we demonstrate the intrinsic ability of non-replicating virions to induce comparable immune responses in the notable absence of any adjuvant. Injection of inactivated pseudorabies virus, an alphaherpesvirus, by various routes into mice resulted in the generation of T helper (Th) 1 type immune response. Co-delivery of inactivated pseudorabies herpesvirus (iPRV) with protein redirected IgG1-dominated tetanus toxoid-specific responses towards an IgG1/IgG2a balanced response. Also inactivated preparations of viruses from the paramyxo- (Newcastle disease virus), rhabdo- (rabies virus), corona- (infectious bronchitis virus) and reovirus (avian reovirus) families led to IgG2a antibody responses; however, the genetic background of the host did result in considerable variation. Because disrupted virions also induced type 1 immune responses, we conclude that structural elements of virions inherently contribute to IFN-gamma-dependent isotype switching by inactivated viruses. Strikingly, immunizations in gene-disrupted mice showed that a functional IFN-alpha/beta, IFN-gamma or interleukin (IL)-12 pathway was not required for the generation of a polarized Th1 type immune response initiated by inactivated virus particles. These findings have a bearing on the understanding of immune responsiveness to virus structures and the design of vaccines containing virus components.

Toroviruses (family Coronaviridae, order Nidovirales) are enveloped, positive-stranded RNA viruses that have been implicated in enteric disease in cattle and possibly in humans. Despite their potential veterinary and clinical relevance, little is known about torovirus epidemiology and molecular genetics. Here, we present the first study into the diversity among toroviruses currently present in European swine and cattle herds. Comparative sequence analysis was performed focusing on the genes for the structural proteins S, M, HE, and N, with fecal specimens serving as sources of viral RNA. Sequence data published for animal and human torovirus variants were included. Four genotypes, displaying 30 to 40% divergence, were readily distinguished, exemplified by bovine torovirus (BToV) Breda, porcine torovirus (PToV) Markelo, equine torovirus Berne, and the putative human torovirus. The ungulate toroviruses apparently display host species preference. In phylogenetic analyses, all PToV variants clustered, while the recent European BToVs mostly resembled the New World BToV variant Breda, identified 19 years ago. However, we found ample evidence for recurring intertypic recombination. All newly characterized BToV variants seem to have arisen from a genetic exchange, during which the 3' end of the HE gene, the N gene, and the 3' nontranslated region of a Breda virus-like parent had been swapped for those of PToV. Moreover, some PToV and BToV variants carried chimeric HE genes, which apparently resulted from recombination events involving hitherto unknown toroviruses. From these observations, the existence of two additional torovirus genotypes can be inferred. Toroviruses may be even more promiscuous than their closest relatives, the coronaviruses and arteriviruses.

Cell-mediated immunity is thought to play a decisive role in protecting cats against feline infectious peritonitis (FIP), a progressive and lethal coronavirus disease. In view of the potential of DNA vaccines to induce cell-mediated responses, their efficacy to induce protective immunity in cats was evaluated. The membrane (M) and nucleocapsid (N) proteins were chosen as antigens, because antibodies to the spike (S) protein of FIP virus (FIPV) are known to precipitate pathogenesis. However, vaccination by repeated injections of plasmids encoding these proteins did not protect kittens against challenge infection with FIPV. Also, a prime-boost protocol failed to afford protection, with priming using plasmid DNA and boosting using recombinant vaccinia viruses expressing the same coronavirus proteins. Because of the role of IL-12 in initiating cell-mediated immunity, the effects of co-delivery of plasmids encoding the feline cytokine were studied. Again, IL-12 did not meet expectations - on the contrary, it enhanced susceptibility to FIPV challenge. This study shows that DNA vaccination failed to protect cats against FIP and that IL-12 may yield adverse effects when used as a cytokine adjuvant.

The envelope is of cardinal importance for the entry of feline immunodeficiency virus (FIV) into its host cells, which consist of cells of the immune system including macrophages. To characterize the envelope glycoprotein determinants involved in macrophage tropism, chimeric infectious molecular clones were constructed containing envelope gene sequences from isolates that had been propagated in peripheral blood mononuclear cells (PBMC). The progeny virus was examined for growth in PBMC and bone marrow-derived macrophages and viruses with different replication kinetics in macrophages were selected. Envelope-chimeric viruses revealed that nucleotide sequences encoding variable regions 3 and 4 of the surface glycoprotein, SU, are involved in macrophage tropism of FIV. To assess the biological importance of this finding, the phenotypes of envelope proteins of viruses derived from bone marrow, brain, lymph node and PBMC of an experimentally FIV-infected, healthy cat were examined. Since selection during propagation had to be avoided, provirus envelope gene sequences were amplified directly and cloned into an infectious molecular clone of FIV strain Petaluma. The viruses obtained were examined for their replication properties. Of 15 clones tested, 13 clones replicated both in PBMC and macrophages, two (brain-derived clones) replicated in PBMC only and none replicated in Crandell feline kidney cells or astrocytes. These results indicate that dual tropism for PBMC and macrophages is a common feature of FIV variants present in vivo.

Bicyclams are low-molecular-weight anti-human immunodeficiency virus (HIV) agents that have been shown to act as potent and selective CXC chemokine receptor 4 (CXCR4) antagonists. Here, we demonstrate that bicyclams are potent inhibitors of feline immunodeficiency virus (FIV) replication when evaluated in Crandell feline kidney (CRFK) cells. With a series of bicyclam derivatives, 50% inhibitory concentrations (IC50s) against FIV were obtained in this cell system that were comparable to those obtained for HIV-1 IIIB replication in the human CD4(+) MT-4 T-cell line. The bicyclams were also able to block FIV replication in feline thymocytes, albeit at higher concentrations than in the CRFK cells. The prototype bicyclam AMD3100, 1-1'-[1,4-phenylene-bis(methylene)]-bis(1,4,8, 11-tetraazacyclotetradecane), was only fourfold less active in feline thymocytes (IC50, 62 ng/ml) than in CRFK cells (IC50, 14 ng/ml). AMD2763, 1,1'-propylene-bis(1,4,8, 11-tetraazacyclotetradecane), which is a less potent CXCR4 antagonist, was virtually inactive against FIV in feline thymocytes (IC50, >66.5 microgram/ml), while it was clearly active in CRFK cells (IC50, 0.9 microgram/ml). The CXC chemokine stromal-cell-derived factor 1alpha had anti-FIV activity in CRFK cells (IC50, 200 ng/ml) but not in feline thymocytes (IC50, >2.5 microgram/ml). When primary FIV isolates were evaluated for their drug susceptibility in feline thymocytes, the bicyclams AMD3100 and its Zn2+ complex, AMD3479, inhibited all six primary isolates at equal potency. The marked susceptibility of FIV to the bicyclams suggests that FIV predominantly uses feline CXCR4 for entering its target cells.

The alphaherpesvirus glycoproteins gE and gI form a hetero-oligomeric complex involved in cell-to-cell transmission. The gI-deficient recombinant feline herpesvirus (FHV), FHVdeltagI-LZ, produces plaques that are only 15% the size of those of wild-type FHV. Here, we have complemented FHV(delta)gI-LZ allotopically by expressing intact gI and C-terminally truncated gI derivatives from the thymidine kinase locus. The effect on gE-gI-mediated cell-to-cell spread was assessed by plaque assay employing computer-assisted image analysis (software available at http://www.androclus.vet.uu.nl/spotter/spotter.htm+ ++). Allotopic complementation with intact gI fully restored plaque size. Deletion of the C-terminal 11 residues of gI did not affect cell-to-cell spread, whereas deletion of the complete cytoplasmic tail reduced plaque size by only 35%. Mutants expressing gI166, roughly corresponding to the N-terminal half of the ectodomain, displayed a small-plaque phenotype. Nevertheless, their plaques were reproducibly larger than those of matched gI-deficient controls, indicating that the gE-gI166 hetero-oligomer, though crippled, is still able to mediate cell-to-cell spread. Our data demonstrate that plaque analysis provides a reliable and convenient tool to measure and quantitate gE-gI function in vitro.

We analysed the ability of a plasmid carrying the gene encoding glycoprotein D (gD) of pseudorabies virus (PRV) to induce humoral and cell-mediated immune responses and assessed the protection provided by PRV-gD DNA vaccination against challenge infection with PRV. Immunization with plasmid PRV-gD induced neutralizing antibodies and lymphocyte proliferative responses both in mice and pigs. Moreover, when challenged with virulent PRV six weeks following the last immunization, PRV-gD DNA vaccinated pigs excreted virus for a significantly shorter period and showed less clinical symptoms than pigs vaccinated with a control plasmid. Thus, in the target animal, DNA vaccination with PRV-gD DNA induces protective immunity against challenge infection.

Adenoviral nucleic acid was detected by polymerase chain reaction (PCR) in pharyngeal and rectal swab samples of a cat seropositive for adenovirus and suffering from transient hepatic failure. The samples were taken at a one-year interval, and both faecal samples as well as the second pharyngeal sample were positive in PCR performed with general adenovirus primers. The size of the amplified products corresponded to that of the positive control. The identity of the amplicons was also confirmed by DNA sequencing. The 301 bp long hexon gene fragment was very similar to but distinguishable from the corresponding hexon sequence of human adenovirus type 2. This result suggests the possibility of persistent carrier status and shedding of adenovirus in cats.

The causative agent of tobacco mosaic and of foot and mouth disease (FMD) were recognized in 1898 as "filterable" or "invisible"--and eventually termed "virus". Four years later the viral aetiology of yellow fever was established, and the new discipline took off. Thus animal virology started with a veterinary problem, and Germany's contribution during the following decades came mainly from the chairs of veterinary teaching and research establishments in Giessen, Munich and Hanover, the Riems Institute, and the Federal Research Institute for Animal Virus Diseases in Tübingen. From a superficial bibliometric analysis, a wide divergence in impact figures is noted, with excellent contributions in international virology journals and lesser papers in German veterinary journals. The publications in the observed time frame reveal a fascination by virion structure, physical characteristics and structure-function relationships with little work published in journals dedicated to immunology and pathogenesis.

Alphaherpesvirus glycoproteins E and I (gE and gI, respectively) assemble into a hetero-oligomeric complex which promotes cell-to-cell transmission, a determining factor of virulence. Focusing on gI of feline herpesvirus (FHV), we examined the role of disulfide bonds during its biosynthesis, its interaction with gE, and gE-gI-mediated spread of the infection in vitro. The protein's disulfide linkage pattern was determined by single and pairwise substitutions for the four conserved cysteine residues in the ectodomain. The resulting mutants were coexpressed with gE in the vaccinia virus-based vTF7-3 system, and the formation and endoplasmic reticulum (ER)-to-Golgi transport of the hetero-oligomeric complex were monitored. The results were corroborated biochemically by performing an endoproteinase Lys-C digestion of a [35S]Cys-labeled secretory recombinant form of gI followed by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the peptides under reducing and nonreducing conditions. We found that (i) gI derivatives lacking Cys79 (C1) and/or Cys223 (C4) still assemble with gE into transport-competent complexes, (ii) mutant proteins lacking Cys91 (C2) and/or Cys102 (C3) bind to gE but are retained in the ER, (iii) radiolabeled endoproteinase Lys-C-generated peptide species containing C1 and C4 are linked through disulfide bonds, and (iv) peptides containing both C2 and C3 are not disulfide linked to any other peptide. From these findings emerges a model in which C1 and C4 as well as C2 and C3 form intramolecular disulfide bridges. Since the cysteines in the ectodomain have been conserved during alphaherpesvirus divergence, we postulate that the model applies for all gI proteins. Analysis of an FHV recombinant with a C1-->S substitution confirmed that the C1-C4 disulfide bond is not essential for the formation of a transport-competent gE-gI complex. The mutation affected the posttranslational modification of gI and caused a slight cold-sensitivity defect in the assembly or the intracellular transport of the gE-gI complex but did not affect plaque size. Thus, C1 and the C1-C4 bond are not essential for gE-gI-mediated cell-to-cell spread, at least not in vitro.

A putative bovine respiratory torovirus (BRTV) was propagated in bovine fetal diploid lung and human colonic tumour cells, and fringed pleomorphic particles were detected in the culture supernatants by electron microscopy. Antisera directed against a bovine (Breda strain) and equine (Berne strain) torovirus failed to react with BRTV-infected cells in immunofluorescence assays and did not neutralise BRTV. No toroviral RNA was found in the supernatants of infected cells by means of a reverse transcriptase-polymerase chain reaction with torovirus-specific primers. On the other hand, bovine coronavirus-specific antisera and monoclonal antibodies did neutralise the cytopathic effects, and coronaviral antigen was detected in the cultures by immunofluorescence. Furthermore, bovine coronavirus RNA was detected in the supernatants of BRTV-infected cells after nucleic acid amplification. It is concluded that the cytopathic BRTV isolate is a coronavirus.

A porcine torovirus (PoTV) was identified and characterized; it is a novel member of the genus Torovirus (family Coronaviridae, order Nidovirales), closely related to but clearly distinct from the already recognized equine torovirus (ETV) and bovine torovirus (BoTV) representatives. Immunoelectron microscopy of feces from piglets revealed elongated, 120- by 55-nm particles which were recognized by a torovirus-specific antiserum. Amplification by reverse transcriptase (RT) PCR with primers designed to detect conserved regions (on the basis of the genomes of BoTV strain Breda and ETV strain Berne) resulted in the identification of the 489-bp nucleocapsid gene, encoding a 18.7-kDa protein. The sequence identity in this region between PoTV and both ETV and BoTV was only about 68%, whereas the latter two show 81% identity. Neutralizing antibodies directed against ETV were found in sera of adult and young pigs. In all 10 herds sampled, seropositive animals were present, and 81% of randomly selected adult sows possessed antibodies. A longitudinal study with RT PCR showed that piglets shed virus in the feces for 1 or more days, starting 4 to 14 days after weaning.

Recent evidence suggests that the type II feline coronavirus (FCoV) strains 79-1146 and 79-1683 have arisen from a homologous RNA recombination event between FCoV type I and canine coronavirus (CCV). In both cases, the template switch apparently took place between the S and M genes, giving rise to recombinant viruses which encode a CCV-like S protein and the M, N, 7a, and 7b proteins of FCoV type I (K. Motowaka, T. Hohdatsu, H. Hashimoto, and H. Koyama, Microbiol. Immunol. 40:425-433, 1996; H. Vennema, A. Poland, K. Floyd Hawkins, and N. C. Pedersen, Feline Pract. 23:40-44, 1995). In the present study, we have looked for additional FCoV-CCV recombination sites. Four regions in the pol gene were selected for comparative sequence analysis of the type II FCoV strains 79-1683 and 79-1146, the type I FCoV strains TN406 and UCD1, the CCV strain K378, and the TGEV strain Purdue. Our data show that the type II FCoVs have arisen from double recombination events: additional crossover sites were mapped in the ORF1ab frameshifting region of strain 79-1683 and in the 5' half of ORF1b of strain 79-1146.

Studies in IL-12-deficient mice established the necessity for IL-12 to generate a Th1 cytokine response that is often required for elimination of intracellular pathogens. In this study, we demonstrate that mice with a targeted disruption of the IL-12p40 and/or p35 gene effectively control liver damage induced by mouse hepatitis virus (MHV) infection, similar to wild-type animals. In contrast, MHV-infected IFN-gamma receptor-deficient (IFN-gammaR[-/-]) mice showed an increased susceptibility to coronaviral hepatitis. Surprisingly, MHV-infected mice lacking IL-12 produced a polarized Th1-type cytokine response, as evidenced by high IFN-gamma and nondetectable IL-4 production by CD4+ splenocytes and normal virus-specific serum IgG2a/IgG1 ratios. The virus-induced type 1 cytokine secretion pattern was not reversed in IL-12-deficient mice by in vivo neutralization of IFN-gamma nor in IFN-gammaR(-/-) mice receiving IL-12-neutralizing Abs. In IL-12-deficient mice, Th1-type responses were also generated upon immunization with inactivated MHV. In contrast, following immunization with keyhole limpet hemocyanin, mice lacking IL-12 mounted strongly reduced specific IgG2a and increased IgE responses, indicative of a type 2-dominated cytokine pattern. These findings demonstrate that following a virus infection, IL-12 is not essential for the generation of polarized T cell type 1 cytokine expression and associated immune responses, which is in marked contrast to nonviral systems. Our data suggest that viruses may selectively induce IFN-gamma production and Th1-type immune reactions even in the absence of IL-12.

Coronaviruses are assembled by budding into a pre-Golgi compartment from which they are transported along the secretory pathway to leave the cell. In cultured epithelial cells, they are released in a polarized fashion; depending on the virus and cell type, they are sorted preferentially either to the apical domain or to the basolateral plasma membrane domain. In this study, we investigated the role of the coronavirus spike protein, because of its prominent position in the virion the prime sorting candidate, in the directionality of virus release. Three independent approaches were taken. (i) The inhibition of N glycosylation by tunicamycin resulted in the synthesis of spikeless virions. The absence of spikes, however, did not influence the polarity in the release of virions. Thus, murine hepatitis virus strain A59 (MHV-A59) was still secreted from the basolateral membranes of mTAL and LMR cells and from the apical sides of MDCK(MHVR) cells, whereas transmissible gastroenteritis virus (TGEV) was still released from the apical surfaces of LMR cells. (ii) Spikeless virions were also studied by using the MHV-A59 temperature-sensitive mutant Albany 18. When these virions were produced in infected LMR and MDCK(MHVR) cells at the nonpermissive temperature, they were again preferentially released from basolateral and apical membranes, respectively. (iii) We recently demonstrated that coronavirus-like particles resembling normal virions were assembled and released when the envelope proteins M and E were coexpressed in cells (H. Vennema, G.-J. Godeke, J. W. A. Rossen, W. F. Voorhout, M. C. Horzinek, D.-J. E. Opstelten, and P. J. M. Rottier, EMBO J. 15:2020-2028, 1996). The spikeless particles produced in mTAL cells by using recombinant Semliki Forest viruses to express these two genes of MHV-A59 were specifically released from basolateral membranes, i.e., with the same polarity as that of wild-type MHV-A59. Our results thus consistently demonstrate that the spike protein is not involved in the directional sorting of coronaviruses in epithelial cells. In addition, our observations with tunicamycin show that contrary to the results with some secretory proteins, the N-linked oligosaccharides present on the viral M proteins of coronaviruses such as TGEV also play no role in viral sorting. The implications of these conclusions are discussed.

Alphaherpesvirus glycoproteins gE and gI form a noncovalently associated hetero-oligomeric complex, which is involved in cell-to-cell spread. In the absence of gI, feline herpesvirus (FHV) gE is transport incompetent and fully retained in the endoplasmic reticulum. Here, we assess the effect of progressive C-terminal truncations of FHV gI on the biosynthesis, intracellular transport, and function of the gE-gI complex. The truncated gI proteins were coexpressed with gE in the vaccinia virus-based vTF7-3 expression system. The results were corroborated and extended by studying FHV recombinants expressing truncated gI derivatives. The following conclusions can be drawn. (i) Deletion of the cytoplasmic tail, the transmembrane region plus the C-terminal half of the ectodomain of gI, does not affect intracellular transport of gE. Apparently, the N-terminal 166 residues of gI constitute a domain involved in gE-gI interaction. (ii) A region mediating stable association with gE is located within the N-terminal 93 residues of gI. (iii) The cytoplasmic domain of gI is not essential for gE-gI-mediated cell-to-cell transmission of FHV, as judged from plaque morphology. Deletion of the cytoplasmic tail of gI reduced plaque size by only 35%. (iv) Recombinants expressing the N-terminal 166 residues of gI display a small-plaque phenotype but produce larger plaques than recombinants with a disrupted gI gene. Thus, a complex consisting of gE and the N-terminal half of the gI ectodomain may retain residual biological activity. The implications of these findings for gE-gI interaction and function are discussed.

The cellular tropism of the feline immunodeficiency virus (FIV) is affected by changes in variable region 3 (V3) of the surface (SU) envelope glycoprotein (Verschoor, E. J., et al., J. Virol. 69:4752-4757, 1995). By using high-dose DNA transfection, an FIV molecular clone with a non-CRFK-tropic V3 acquired the ability to replicate in CRFK cells. A single point mutation from a methionine to a threonine in the ectodomain of its transmembrane (TM) envelope glycoprotein was responsible for this change in viral tropism. This substitution is located in the putative SU interactive region, between the fusion peptide and the membrane-spanning region. Our results show that this region of the TM envelope glycoprotein constitutes an additional determinant for cell tropism.

Feline coronavirus (FCoV) persistence and evolution were studied in a closed cat-breeding facility with an endemic serotype I FCoV infection. Viral RNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR) in the feces and/or plasma of 36 of 42 cats (86%) tested. Of 5 cats, identified as FCoV shedders during the initial survey, 4 had detectable viral RNA in the feces when tested 111 days later. To determine whether this was due to continuous reinfection or to viral persistence, 2 cats were placed in strict isolation and virus shedding in the feces was monitored every 2-4 days. In 1 of the cats, virus shedding continued for up to 7 months. The other animal was sacrificed after 124 days of continuous virus shedding in order to identify the sites of viral replication. Viral mRNA was detected only in the ileum, colon, and rectum. Also in these tissues, FCoV-infected cells were identified by immunohistochemistry. These findings provide the first formal evidence that FCoV causes chronic enteric infections. To assess FCoV heterogeneity in the breeding facility and to study viral evolution during chronic infection, FCoV quasispecies sampled from individual cats were characterized by RT-PCR amplification of selected regions of the viral genome followed by sequence analysis. Phylogenetic comparison of nucleotides 7-146 of ORF7b to corresponding sequences obtained for independent European and American isolates indicated that the viruses in the breeding facility form a clade and are likely to have originated from a single founder infection. Comparative consensus sequence analysis of the more variable region formed by residues 79-478 of the S gene revealed that each cat harbored a distinct FCoV quasispecies. Moreover, FCoV appeared to be subject to immune selection during chronic infection. The combined data support a model in which the endemic infection is maintained by chronically infected carriers. Virtually every cat born to the breeding facility becomes infected, indicating that FCoV is spread very efficiently. FCoV-infected cats, however, appear to resist superinfection by closely related FCoVs.

A modified live virus vaccine against feline infectious peritonitis (FIP) was evaluated in a double blind, placebo-controlled field trial in two high-risk populations. The vaccine was found to be safe and efficacious in one population of cats that had low antibody titre against feline coronavirus (FCoV) at the time of vaccination. Although clinically healthy at the time of vaccination, retrospectively some vaccinees that later came down with FIP were found to be RT-PCR positive for FCoV in plasma and showed changes in blood parameters consistent with early stage of FIP. It is concluded that vaccination can protect cats with no or low FCoV antibody titres and that in some cats vaccine failure was probably due to pre-existing infection.

We have characterized the 3'-most 3 kb of the genome of bovine torovirus (BoTV) strain Breda. A novel 1.2-kb gene, located between the genes for the membrane and nucleocapsid proteins, was identified. This gene, the 3'-most 0.5 kb of which is also present in the genome of the equine torovirus isolate Berne virus (BEV), codes for a class I membrane protein displaying 30% sequence identity with the hemagglutinin-esterases (HEs) of coronaviruses and influenza C viruses. Heterologous expression of the BoTV HE gene yielded a 65,000-molecular weight N-glycosylated protein displaying acetylesterase activity. Serologic evidence indicates that the HE homolog is expressed during the natural infection and represents a prominent antigen. By using an antiserum raised against residues 13 to 130 of HE, the HE protein was detected in radioiodinated, sucrose gradient-purified BoTV preparations. Formal evidence that HE is a structural protein was provided by immunoelectron microscopy. In addition to the large, 17- to 20-nm spikes, BoTV virions possess shorter surface projections (6 nm on average). We postulate that these surface projections, which are absent from the BEV virion, are composed of the BoTV HE homolog. The HE gene, which has now been demonstrated in three different virus genera, is a showpiece example of modular evolution.