Canine immunoglobulin F(ab')2 fragments protect cats against feline parvovirus virus infection.

Feline parvovirus virus (FPV) causes severe diarrhea and leukopenia in felines, and threatening the health of wild and domestic felines. Currently, specific drugs to treat FPV have not yet been developed. In this study, IgG was extracted from inactivated FPV-immunized dog sera. Canine F(ab')2 fragments were obtained from pepsin-digested IgG and then purified by protein-G column chromatography. The results showed that canine immunoglobulin F(ab')2 fragments showed efficient neutralizing activity in vitro against FPV and had therapeutic and prophylactic effects in FPV infected cats. The anti-FPV-specific F(ab')2 fragment can significantly alleviate the clinical symptoms of FPV infected cats and reduce the viral loads of the intestinal tract. These results indicated that the F(ab')2 fragment prepared from inactivated FPV-immunized felines may be used as a prophylactic and therapeutic agent for diseases caused by FPV.

New genotype classification and molecular characterization of canine and feline parvoviruses.

BACKGROUND: Canine parvovirus (CPV) and feline panleukopenia (FPV) cause severe intestinal disease and leukopenia. OBJECTIVES: In Korea, there have been a few studies on Korean FPV and CPV-2 strains. We attempted to investigate several genetic properties of FPV and CPV-2. METHODS: Several FPV and CPV sequences from around world were analyzed by Bayesian phylo-geographical analysis. RESULTS: The parvoviruses strains were newly classified into FPV, CPV 2-I, CPV 2-II, and CPV 2-III genotypes. In the strains isolated in this study, Gigucheon, Rara and Jun belong to the FPV, while Rachi strain belong to CPV 2-III. With respect to CPV type 2, the new genotypes are inconsistent with the previous genotype classifications (CPV-2a, -2b, and -2c). The root of CPV-I strains were inferred to be originated from a USA strain, while the CPV-II and III were derived from Italy strains that originated in the USA. Based on VP2 protein analysis, CPV 2-I included CPV-2a-like isolates only, as differentiated by the change in residue S297A/N. Almost CPV-2a isolates were classified into CPV 2-III, and a large portion of CPV-2c isolates was classified into CPV 2-II. Two residue substitutions F267Y and Y324I of the VP2 protein were characterized in the isolates of CPV 2-III only. CONCLUSIONS: We provided an updated insight on FPV and CPV-2 genotypes by molecular-based and our findings demonstrate the genetic characterization according to the new genotypes.

Treatment with Class A CpG Oligodeoxynucleotides in Cats with Naturally Occurring Feline Parvovirus Infection: A Prospective Study.

Feline parvovirus (FPV) causes severe gastroenteritis and leukopenia in cats; the outcome is poor. Information regarding specific treatments is lacking. Class A CpG oligodeoxynucleotides (CpG-A) are short single-stranded DNAs, stimulating type I interferon production. In cats, CpG-A induced an antiviral response in vivo and inhibited FPV replication in vitro. The aim was to prospectively investigate the effects of CpG-A on survival, clinical score, hematological findings, antiviral response (cytokines), viremia, and fecal shedding (real-time qPCR) in cats naturally infected with FPV. Forty-two FPV-infected cats were randomized to receive 100 µg/kg of CpG-A (n = 22) or placebo (n = 20) subcutaneously, on admission and after 48 h. Blood and fecal samples were collected on admission, after 1, 3, and 7 days. All 22 cats showed short duration pain during CpG-A injections. The survival rate, clinical score, leukocyte and erythrocyte counts, viremia, and fecal shedding at any time-point did not differ between cats treated with CpG-A (50%) and placebo (40%). Antiviral myxovirus resistance (Mx) gene transcription increased in both groups from day 1 to 3 (p = 0.005). Antibodies against FPV on admission were associated with survival in cats (p = 0.002). In conclusion, CpG-A treatment did not improve the outcome in cats with FPV infection. FPV infection produced an antiviral response.

Cellular microRNA, miR-1343-5p, modulates IFN-I responses to facilitate feline panleukopenia virus replication by directly targeting IRAK1 gene.

Feline panleukopenia is an acute, highly contagious, and fatal infectious disease caused by feline panleukopenia virus (FPV) and has led to severe consequences on pets, economically important animals, and the wildlife industry. MicroRNAs (miRNAs) play significant roles in the host-pathogen interaction by modulating cellular factors expression which are essential for viral replication or host innate immune response to infection. However, the role of host miRNA response in FPV infection remains to be discovered. In this study, we screened nine host miRNAs associated with FPV infection that were previously implicated in innate immunity or antiviral functions. We found that miR-1343-5p overexpression strongly promoted FPV-BJ04 genomic DNA. Subsequently, the expression of host miR-1343-5p was upregulated by FPV-BJ04 infection in vitro and in vivo. In addition, we demonstrated that miR-1343-5p was a negative regulator of the IFN-I signaling pathway, thereby promoting FPV infection. Bioinformatic analysis combined with molecular biological assay indicated that interleukin-1 receptor-associated kinase 1 (IRAK1) is a putative target of miR-1343-5p. Collectively, our findings emphasize the importance of miR-1343-5p in host defense against FPV, thus, enhancing our understanding of its pathogenic mechanism.

Faecal shedding of parvovirus deoxyribonucleic acid following modified live feline panleucopenia virus vaccination in healthy cats.

Positive canine parvovirus (CPV) faecal test results have been reported in dogs after modified live virus (MLV) vaccination. Thus, the aim was to investigate feline panleucopenia virus (FPV) shedding in recently vaccinated, adult, clinically healthy cats and to assess related factors. Forty cats were vaccinated with an FPV MLV vaccine. Faeces of cats were tested for presence of parvovirus DNA on days 7, 14, 21 and 28 by quantitative real-time PCR; DNA-positive samples were subjected to partial VP2 gene sequencing. Virus isolation was performed whenever sufficient amounts of faeces were available. Serum antibody titres were measured by haemagglutination inhibition on days 0, 7 and 28. Overall, 30.0 per cent (12/40; 95% CI 18.0 to 45.6) of cats shed parvovirus DNA. Sequencing revealed FPV vaccine virus DNA in three cats, FPV field virus DNA in four cats and CPV field virus DNA in one cat. Shedding was significantly associated with lack of prevaccination antibody titres (40) (P=0.016; OR: 6.44; 95% CI 1.44 to 28.89) and with postvaccination titre increases (fourfold) (P=0.029; OR: 5.00; 95% CI 1.17 to 21.39). Shedding of field or vaccine virus DNA seems to be common in healthy cats which can be a concern in shelters and catteries. Diagnostic tools should be developed to facilitate differentiation of vaccine and field virus shedding.

Carnivore Parvovirus Ecology in the Serengeti Ecosystem: Vaccine Strains Circulating and New Host Species Identified.

Carnivore parvoviruses infect wild and domestic carnivores, and cross-species transmission is believed to occur. However, viral dynamics are not well understood, nor are the consequences for wild carnivore populations of the introduction of new strains into wild ecosystems. To clarify the ecology of these viruses in a multihost system such as the Serengeti ecosystem and identify potential threats for wildlife conservation, we analyzed, through real-time PCR, 152 samples belonging to 14 wild carnivore species and 62 samples from healthy domestic dogs. We detected parvovirus DNA in several wildlife tissues. Of the wild carnivore and domestic dog samples tested, 13% and 43%, respectively, were positive for carnivore parvovirus infection, but little evidence of transmission between the wild and domestic carnivores was detected. Instead, we describe two different epidemiological scenarios with separate routes of transmission: first, an endemic feline parvovirus (FPV) route of transmission maintained by wild carnivores inside the Serengeti National Park (SNP) and, second, a canine parvovirus (CPV) route of transmission among domestic dogs living around the periphery of the SNP. Twelve FPV sequences were characterized; new host-virus associations involving wild dogs, jackals, and hyenas were discovered; and our results suggest that mutations in the fragment of the vp2 gene were not required for infection of different carnivore species. In domestic dogs, 6 sequences belonged to the CPV-2a strain, while 11 belonged to the CPV-2 vaccine-derived strain. This is the first description of a vaccine-derived parvovirus strain being transmitted naturally.IMPORTANCE Carnivore parvoviruses are widespread among wild and domestic carnivores, which are vulnerable to severe disease under certain circumstances. This study furthers the understanding of carnivore parvovirus epidemiology, suggesting that feline parvoviruses are endemic in wild carnivores in the Serengeti National Park (SNP), with new host species identified, and that canine parvoviruses are present in the dog population living around the SNP. Little evidence of transmission of canine parvoviruses into wild carnivore species was found; however, the detection of vaccine-derived virus (described here for the first time to be circulating naturally in domestic dogs) highlights the importance of performing epidemiological research in the region.

Cell cycle S phase markers are expressed in cerebral neuron nuclei of cats infected by the Feline Panleukopenia Virus.

The cell cycle-associated neuronal death hypothesis, which has been proposed as a common mechanism for most neurodegenerative diseases, is notably supported by evidencing cell cycle effectors in neurons. However, in naturally occurring nervous system diseases, these markers are not expressed in neuron nuclei but in cytoplasmic compartments. In other respects, the Feline Panleukopenia Virus (FPV) is able to complete its cycle in mature brain neurons in the feline species. As a parvovirus, the FPV is strictly dependent on its host cell reaching the cell cycle S phase to start its multiplication. In this retrospective study on the whole brain of 12 cats with naturally-occurring, FPV-associated cerebellar atrophy, VP2 capsid protein expression was detected by immunostaining not only in some brain neuronal nuclei but also in neuronal cytoplasm in 2 cats, suggesting that viral mRNA translation was still occurring. In these cats, double immunostainings demonstrated the expression of cell cycle S phase markers cyclin A, cdk2 and PCNA in neuronal nuclei. Parvoviruses are able to maintain their host cells in S phase by triggering the DNA damage response. S139 phospho H2A1, a key player in the cell cycle arrest, was detected in some neuronal nuclei, supporting that infected neurons were also blocked into the S phase. PCR studies did not support a co-infection with an adeno or herpes virus. ERK1/2 nuclear accumulation was observed in some neurons suggesting that the ERK signaling pathway might be involved as a mechanism driving these neurons far into the cell cycle.

Feline parvovirus infection and associated diseases.

Feline panleukopenia, caused by the single-stranded DNA virus feline parvovirus (FPV), is a highly contagious and often lethal disease of cats and other Felidae. FPV, but also canine parvovirus (CPV) can be isolated from both healthy and diseased cats. In Germany, CPV was detected in only approximately 10% of feline samples, but in Southeast Asia, reports estimated that up to approximately 80% of diseased cats were infected with CPV. Infection spreads rapidly, especially in cells with high mitotic activity, such as bone marrow, lymphoid tissue and intestinal crypt cells. Anorexia, vomiting, diarrhoea, neutropenia and lymphopenia are common in clinically affected cases. In utero or neonatal infection can result in cerebellar hypoplasia. Depending on the severity of clinical signs, mortality ranges from 25 to 100%. Effective vaccination and thorough disinfection are of the utmost importance in the prevention of disease transmission in multi-cat households and animal shelters. If clinical signs develop, supportive treatment should be commenced. The efficacy of feline recombinant interferon and FPV antibodies has not been clearly demonstrated. Commercially available vaccines should induce protective immunity when administered according to current guidelines. Recent studies suggest that in some kittens, maternally derived antibodies (MDA) can persist for much longer than has been previously recognised. FPV serum antibody tests are available, but protection status needs to be interpreted with caution in kittens with MDA and a negative titre in adult cats does not necessarily denote lack of protection.

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

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.

Binding site on the transferrin receptor for the parvovirus capsid and effects of altered affinity on cell uptake and infection.

Canine parvovirus (CPV) and its relative feline panleukopenia virus (FPV) bind the transferrin receptor type 1 (TfR) to infect their host cells but show differences in the interactions with the feline and canine TfRs that determine viral host range and tissue tropism. We changed apical and protease-like domain residues by introducing point mutations and adding or removing glycosylation signals, and we then examined the interactions of those mutant TfRs with the capsids. Most substitutions had little effect on virus binding and uptake. However, mutations of several sites in the apical domain of the receptor either prevented binding to the capsids or reduced the affinity of receptor binding to various degrees. Glycans within the virus binding face of the apical domain also controlled capsid binding. CPV, but not the related feline parvovirus, could use receptors containing a canine TfR-specific glycosylation to mediate efficient infection, while addition of other N-linked glycosylation sites into the virus binding face of the feline apical domain reduced or eliminated both binding and infection. Replacement of critical feline TfR residue 221 with every amino acid had effects on binding and infection which were significantly associated with the biochemical properties of the residue replaced. Receptors with reduced affinities mostly showed proportional changes in their ability to mediate infection. Testing feline TfR variants for their binding and uptake patterns in cells showed that low-affinity versions bound fewer capsids and also differed in attachment to the cell surface and filopodia, but transport to the perinuclear endosome was similar.

Activation of p38 MAPK by feline infectious peritonitis virus regulates pro-inflammatory cytokine production in primary blood-derived feline mononuclear cells.

Feline infectious peritonitis (FIP) is an invariably fatal disease of cats caused by systemic infection with a feline coronavirus (FCoV) termed feline infectious peritonitis virus (FIPV). The lethal pathology associated with FIP (granulomatous inflammation and T-cell lymphopenia) is thought to be mediated by aberrant modulation of the immune system due to infection of cells such as monocytes and macrophages. Overproduction of pro-inflammatory cytokines occurs in cats with FIP, and has been suggested to play a significant role in the disease process. However, the mechanism underlying this process remains unknown. Here we show that infection of primary blood-derived feline mononuclear cells by FIPV WSU 79-1146 and FIPV-DF2 leads to rapid activation of the p38 MAPK pathway and that this activation regulates production of the pro-inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta). FIPV-induced p38 MAPK activation and pro-inflammatory cytokine production was inhibited by the pyridinyl imidazole inhibitors SB 203580 and SC 409 in a dose-dependent manner. FIPV-induced p38 MAPK activation was observed in primary feline blood-derived mononuclear cells individually purified from multiple SPF cats, as was the inhibition of TNF-alpha production by pyridinyl imidazole inhibitors.

Parvovirus infection of cells by using variants of the feline transferrin receptor altering clathrin-mediated endocytosis, membrane domain localization, and capsid-binding domains.

The feline and canine transferrin receptors (TfRs) bind canine parvovirus to host cells and mediate rapid capsid uptake and infection. The TfR and its ligand transferrin have well-described pathways of endocytosis and recycling. Here we tested several receptor-dependent steps in infection for their role in virus infection of cells. Deletions of cytoplasmic sequences or mutations of the Tyr-Thr-Arg-Phe internalization motif reduced the rate of receptor uptake from the cell surface, while polar residues introduced into the transmembrane sequence resulted in increased degradation of transferrin. However, the mutant receptors still mediated efficient virus infection. In contrast, replacing the cytoplasmic and transmembrane sequences of the feline TfR with those of the influenza virus neuraminidase (NA) resulted in a receptor that bound and endocytosed the capsid but did not mediate viral infection. This chimeric receptor became localized to detergent-insoluble membrane domains. To test the effect of structural virus receptor interaction on infection, two chimeric receptors were prepared which contained antibody-variable domains that bound the capsid in place of the TfR ectodomain. These chimeric receptors bound CPV capsids and mediated uptake but did not result in cell infection. Adding soluble feline TfR ectodomain to the virus during that uptake did not allow infection.

[Evolution and host variation of the canine parvovirus: molecular basis for the development of a new virus]. / Evolution und Wirtswechsel des Caninen Parvovirus: Molekulare Grundlagen der Entstehung eines neuen Virus.

Canine parvovirus (CPV) evolved as a new pathogen in dogs between 1976 and 1978 from feline panleukopenia virus (FPV). The new virus hit an unprotected population, caused a dramatic pandemic and infected virtually all populations of domestic and wild carnivores worldwide. The great similarity between the two viruses and their differences in host range, both in vivo as well as in vitro, make it a good model system for emerging diseases and host range shifts of viruses. Recent results showed that CPV expanded its host range by binding to the canine transferrin receptor (Tfr). Residues in the capsid protein that had been defined as host range controlling regions also control the binding to the canine transferrin receptor. These residues are located on a raised region of the capsid at the three-fold axis of symmetry. Interestingly, adaption of the new virus to the new host appears to correlate with an improved binding to the Tfr receptor.

Structures of host range-controlling regions of the capsids of canine and feline parvoviruses and mutants.

Canine parvovirus (CPV) and feline panleukopenia virus (FPV) differ in their ability to infect dogs and dog cells. Canine cell infection is a specific property of CPV and depends on the ability of the virus to bind the canine transferrin receptor (TfR), as well as other unidentified factors. Three regions in the capsid structure, located around VP2 residues 93, 300, and 323, can all influence canine TfR binding and canine cell infection. These regions were compared in the CPV and FPV capsid structures that have been determined, as well as in two new structures of CPV capsids that contain substitutions of the VP2 Asn-93 to Asp and Arg, respectively. The new structures, determined by X-ray crystallography to 3.2 and 3.3 A resolutions, respectively, clearly showed differences in the interactions of residue 93 with an adjacent loop on the capsid surface. Each of the three regions show small differences in structure, but each appears to be structurally independent of the others, and the changes likely act together to affect the ability of the capsid to bind the canine TfR and to infect canine cells. This emphasizes the complex nature of capsid alterations that change the virus-cell interaction to allow infection of cells from different hosts.

Combinations of two capsid regions controlling canine host range determine canine transferrin receptor binding by canine and feline parvoviruses.

Feline panleukopenia virus (FPV) and its host range variant, canine parvovirus (CPV), can bind the feline transferrin receptor (TfR), while only CPV binds to the canine TfR. Introducing two CPV-specific changes into FPV (at VP2 residues 93 and 323) endowed that virus with the canine TfR binding property and allowed canine cell infection, although neither change alone altered either property. In CPV the reciprocal changes of VP2 residue 93 or 323 to the FPV sequences individually resulted in modest reductions in infectivity for canine cells. Changing both residues in CPV to the FPV amino acids blocked the canine cell infection, but that virus was still able to bind the canine TfR at low levels. This shows that both CPV-specific changes control canine TfR binding but that binding is not always sufficient to mediate infection.

Residues in the apical domain of the feline and canine transferrin receptors control host-specific binding and cell infection of canine and feline parvoviruses.

Canine parvovirus (CPV) and feline panleukopenia virus (FPV) capsids bind to the transferrin receptors (TfRs) of their hosts and use these receptors to infect cells. The binding is partially host specific, as FPV binds only to the feline TfR, while CPV binds to both the canine and feline TfRs. The host-specific binding is controlled by a combination of residues within a raised region of the capsid. To define the TfR structures that interact with the virus, we altered the apical domain of the feline or canine TfR or prepared chimeras of these receptors and tested the altered receptors for binding to FPV or CPV capsids. Most changes in the apical domain of the feline TfR did not affect binding, but replacing Leu221 with Ser or Asp prevented receptor binding to either FPV or CPV capsids, while replacing Leu221 with Lys resulted in a receptor that bound only to CPV but not to FPV. Analysis of recombinants of the feline and canine TfRs showed that sequences controlling CPV-specific binding were within the apical domain and that more than one difference between these receptors determined the CPV-specific binding of the canine TfR. Single changes within the canine TfR which removed a single amino acid insertion or which eliminated a glycosylation site gave that receptor the expanded ability to bind to FPV and CPV. In some cases, binding of capsids to mutant receptors did not result in infection, suggesting a structural role for the receptor in cell infection by the viruses.

The natural host range shift and subsequent evolution of canine parvovirus resulted from virus-specific binding to the canine transferrin receptor.

Canine parvovirus (CPV) is a host range variant of a feline virus that acquired the ability to infect dogs through changes in its capsid protein. Canine and feline viruses both use the feline transferrin receptor (TfR) to infect feline cells, and here we show that CPV infects canine cells through its ability to specifically bind the canine TfR. Receptor binding on host cells at 37 degrees C only partially correlated with the host ranges of the viruses, and an intermediate virus strain (CPV type 2) bound to higher levels on cells than did either the feline panleukopenia virus or a later strain of CPV. During the process of adaptation to dogs the later variant strain of CPV gained the ability to more efficiently use the canine TfR for infection and also showed reduced binding to feline and canine cells compared to CPV type 2. Differences on the top and the side of the threefold spike of the capsid surface controlled specific TfR binding and the efficiency of binding to feline and canine cells, and these differences also determined the cell infection properties of the viruses.

Feline host range of canine parvovirus: recent emergence of new antigenic types in cats.

Since the emergence of Canine parvovirus (CPV-2) in the late 1970s, CPV-2 has evolved consecutively new antigenic types, CPV-2a and 2b. Although CPV-2 did not have a feline host range, CPV-2a and 2b appear to have gained the ability to replicate in cats. Recent investigations demonstrate the prevalence of CPV-2a and 2b infection in a wide range of cat populations. We illustrate the pathogenic potential of CPV in cats and assess the risk caused by CPV variants.

A heterogeneous nuclear ribonucleoprotein A/B-related protein binds to single-stranded DNA near the 5' end or within the genome of feline parvovirus and can modify virus replication.

Phage display of cDNA clones prepared from feline cells was used to identify host cell proteins that bound to DNA-containing feline panleukopenia virus (FPV) capsids but not to empty capsids. One gene found in several clones encoded a heterogeneous nuclear ribonucleoprotein (hnRNP)-related protein (DBP40) that was very similar in sequence to the A/B-type hnRNP proteins. DBP40 bound specifically to oligonucleotides representing a sequence near the 5' end of the genome which is exposed on the outside of the full capsid but did not bind most other terminal sequences. Adding purified DBP40 to an in vitro fill-in reaction using viral DNA as a template inhibited the production of the second strand after nucleotide (nt) 289 but prior to nt 469. DBP40 bound to various regions of the viral genome, including a region between nt 295 and 330 of the viral genome which has been associated with transcriptional attenuation of the parvovirus minute virus of mice, which is mediated by a stem-loop structure of the DNA and cellular proteins. Overexpression of the protein in feline cells from a plasmid vector made them largely resistant to FPV infection. Mutagenesis of the protein binding site within the 5' end viral genome did not affect replication of the virus.

New quantitative methods for detection of feline parvovirus (FPV) and virus neutralizing antibody against FPV using a feline T lymphoid cell line.

Previously, we reported that a feline T lymphoid cell line, FL74 cells, was very sensitive to feline parvovirus (FPV) infection. In the present study, we developed new quantitative methods for detection of FPV and virus neutralizing antibody against FPV using FL74 cells. The methods presented here were very simple and applicable to both canine parvovirus and feline panleukopenia virus.