Masami Mochizuki

Antigenic and genomic variabilities among recently prevalent parvoviruses of canine and feline origin in Japan.

Canine parvovirus type 2 (CPV-2) and feline panleukopenia (FLP) virus (FPLV) are well known and ubiquitous diarrhea-causing pantropic viruses. A new antigenic variant of CPV-2 (designated as CPV-2a) has been also prevalent among dogs in Japan. In the present study, 24 canine and 8 feline isolates collected during 1987-1991 were compared with 17 CPV-2 or CPV-2a and 7 FPLV strains that had been characterized previously. Genomic properties were determined by the restriction cleavage patterns of amplified genes encoding the capsid proteins VP1 and VP2 by the polymerase chain reaction. Antigenic properties were determined by hemagglutination-inhibition assay with monoclonal antibodies against an FPLV strain. Growth characteristics in feline CRFK and canine MDCK cells were also examined. Genomic and antigenic properties of the canine isolates were relatively invariable with one exceptional isolate, C27, which was recovered from a typical clinical case of parvovirus infection but possessed properties similar to FPLV rather than CPV-2 and CPV-2a. All isolates from FPL cases possessed the same genomic and antigenic properties as those of reference FPLVs isolated in the 1970s, but three of five strains isolated from the feces of clinically healthy cats were likely to be of canine origin because they possessed very similar properties to CPV-2a. Although species-specificity of these novel isolates could not be determined definitely, the results indicate a possibility that transmission of parvovirus has occurred between these two animal species.

Inhibitory effects of recombinant feline interferon on the replication of feline enteropathogenic viruses in vitro

n Abstractn n Antiviral activities of a recombinant feline interferon (rFeIFN) KT-80 were evaluated against feline enteropathogenic viruses in feline and canine cell lines. Sensitivity to antiviral activities of the rFeIFN varied with cell types; Felis catus whole fetus (fcwf-4) cells were more sensitive than Crandell feline kidney cells, but no sensitivity was found for Madin-Darby canine kidney cells when vesicular stomatitis virus was used as a challenge virus. Reductions were generally IFN dose-dependent and were more consistent when the cells were continuously treated with the rFeIFN than when they were pretreated only before viral challenge. Compared with each virus control culture of fcwf-4 cells, yields of rotavirus, feline panleukopenia virus (FPLV), feline calicivirus and feline infectious peritonitis coronavirus were reduced by ranges of 1.3 to ⩽3.1 log10, 0.6 log2, 0.8 to 3.7 log10 and 0.5 to 0.6 log10, respectively, in the cultures continuously treated with 10 to 10000 U of the rFeIFN. The yield reduction of FPLV was considered to be in part attributable to inhibition of cell growth by the rFeIFN supplemented in the medium.n n

Hemagglutinin activity of two distinct genogroups of feline and canine rotavirus strains

SummaryGenomes of hemagglutinating strains of feline and canine rotaviruses, were much more closely related to each other than to non-hemagglutinating strains. The Cat2 feline rotavirus appears to derive from reassortment between hemagglutinating and non-hemagglutinating strains.

Multiple ferritins are vital to successful blood feeding and reproduction of the hard tick Haemaphysalis longicornis

SUMMARY Ticks are obligate hematophagous parasites and important vectors of diseases. The large amount of blood they consume contains great quantities of iron, an essential but also toxic element. The function of ferritin, an iron storage protein, and iron metabolism in ticks need to be further elucidated. Here, we investigated the function a newly identified secreted ferritin from the hard tick Haemaphysalis longicornis (HlFER2), together with the previously identified intracellular ferritin (HlFER1). Recombinant ferritins, expressed in Escherichia coli, were used for anti-serum preparation and were also assayed for iron-binding activity. RT-PCR and western blot analyses of different organs and developmental stages of the tick during blood feeding were performed. The localization of ferritins in different organs was demonstrated through an indirect immunofluorescent antibody test. RNA interference (RNAi) was performed to evaluate the importance of ferritin in blood feeding and reproduction of ticks. The midgut was also examined after RNAi using light and transmission electron microscopy. RT-PCR showed differences in gene expression in some organs and developmental stages. Interestingly, only HlFER2 was detected in the ovary during oviposition and in the egg despite the low mRNA transcript. RNAi induced a reduction in post-blood meal body weight, high mortality and decreased fecundity. The expression of vitellogenin genes was affected by silencing of ferritin. Abnormalities in digestive cells, including disrupted microvilli, and alteration of digestive activity were also observed. Taken altogether, our results show that the iron storage and protective functions of ferritin are crucial to successful blood feeding and reproduction of H. longicornis.

Emergence of pathogenic coronaviruses in cats by homologous recombination between feline and canine coronaviruses.

Type II feline coronavirus (FCoV) emerged via double recombination between type I FCoV and type II canine coronavirus (CCoV). In this study, two type I FCoVs, three type II FCoVs and ten type II CCoVs were genetically compared. The results showed that three Japanese type II FCoVs, M91-267, KUK-H/L and Tokyo/cat/130627, also emerged by homologous recombination between type I FCoV and type II CCoV and their parent viruses were genetically different from one another. In addition, the 3′-terminal recombination sites of M91-267, KUK-H/L and Tokyo/cat/130627 were different from one another within the genes encoding membrane and spike proteins, and the 5′-terminal recombination sites were also located at different regions of ORF1. These results indicate that at least three Japanese type II FCoVs emerged independently. Sera from a cat experimentally infected with type I FCoV was unable to neutralize type II CCoV infection, indicating that cats persistently infected with type I FCoV may be superinfected with type II CCoV. Our previous study reported that few Japanese cats have antibody against type II FCoV. All of these observations suggest that type II FCoV emerged inside the cat body and is unable to readily spread among cats, indicating that these recombination events for emergence of pathogenic coronaviruses occur frequently.

Characterization of a canine rotavirus strain by neutralization and molecular hybridization assays.

SummaryA canine rotavirus, RS-15, previously isolated in Japan was determined to be subgroup I and serotype 3. When compared with prototype human and animal rotavirus strains by RNA-RNA hybridization assay, the RS-15 strain showed a high degree of homology only with the canine CU-1 strain isolated in the United States of America, suggesting that canine rotaviruses constitute a distinct gene family, which we have elsewhere proposed to term “genogroup”.

Two Kinds of Ferritin Protect Ixodid Ticks from Iron Overload and Consequent Oxidative Stress

Ticks are obligate hematophagous parasites that have successfully developed counteractive means against their hosts immune and hemostatic mechanisms, but their ability to cope with potentially toxic molecules in the blood remains unclear. Iron is important in various physiological processes but can be toxic to living cells when in excess. We previously reported that the hard tick Haemaphysalis longicornis has an intracellular (HlFER1) and a secretory (HlFER2) ferritin, and both are crucial in successful blood feeding and reproduction. Ferritin gene silencing by RNA interference caused reduced feeding capacity, low body weight and high mortality after blood meal, decreased fecundity and morphological abnormalities in the midgut cells. Similar findings were also previously reported after silencing of ferritin genes in another hard tick, Ixodes ricinus. Here we demonstrated the role of ferritin in protecting the hard ticks from oxidative stress. Evaluation of oxidative stress in Hlfer-silenced ticks was performed after blood feeding or injection of ferric ammonium citrate (FAC) through detection of the lipid peroxidation product, malondialdehyde (MDA) and protein oxidation product, protein carbonyl. FAC injection in Hlfer-silenced ticks resulted in high mortality. Higher levels of MDA and protein carbonyl were detected in Hlfer-silenced ticks compared to Luciferase-injected (control) ticks both after blood feeding and FAC injection. Ferric iron accumulation demonstrated by increased staining on native HlFER was observed from 72 h after iron injection in both the whole tick and the midgut. Furthermore, weak iron staining was observed after Hlfer knockdown. Taken together, these results show that tick ferritins are crucial antioxidant molecules that protect the hard tick from iron-mediated oxidative stress during blood feeding.

Oncolytic Reovirus in Canine Mast Cell Tumor

The usage of reovirus has reached phase II and III clinical trials in human cancers. However, this is the first study to report the oncolytic effects of reovirus in veterinary oncology, focusing on canine mast cell tumor (MCT), the most common cutaneous tumor in dogs. As human and canine cancers share many similarities, we hypothesized that the oncolytic effects of reovirus can be exploited in canine cancers. The objective of this study was to determine the oncolytic effects of reovirus in canine MCT in vitro, in vivo and ex vivo. We demonstrated that MCT cell lines were highly susceptible to reovirus as indicated by marked cell death, high production of progeny virus and virus replication. Reovirus induced apoptosis in the canine MCT cell lines with no correlation to their Ras activation status. In vivo studies were conducted using unilateral and bilateral subcutaneous MCT xenograft models with a single intratumoral reovirus treatment and apparent reduction of tumor mass was exhibited. Furthermore, cell death was induced by reovirus in primary canine MCT samples in vitro. However, canine and murine bone marrow-derived mast cells (BMCMC) were also susceptible to reovirus. The combination of these results supports the potential value of reovirus as a therapy in canine MCT but warrants further investigation on the determinants of reovirus susceptibility.

Iron metabolism in hard ticks (Acari: Ixodidae): The antidote to their toxic diet

Ticks are notorious parasitic arthropods, known for their completely host-blood-dependent lifestyle. Hard ticks (Acari: Ixodidae) feed on their hosts for several days and can ingest blood more than a hundred times their unfed weight. Their blood-feeding habit facilitates the transmission of various pathogens. It is remarkable how hard ticks cope with the toxic nature of their blood meal, which contains several molecules that can promote oxidative stress including iron. While it is required in several physiological processes, high amounts of iron can be dangerous because iron can also participate in the formation of free radicals that may cause cellular damage and death. Here we review the current knowledge on heme and inorganic iron metabolism in hard ticks and compare it with that in vertebrates and other arthropods. We briefly discuss the studies on heme transport, storage and detoxification, and the transport and storage of inorganic iron, with emphasis on the functions of tick ferritins. This review points out other aspects of tick iron metabolism that warrant further investigation, as compared to mammals and other arthropods. Further understanding of this physiological process may help in formulating new control strategies for tick infestation and the spread of tick-borne diseases.

Evaluation and comparison of the potential of two ferritins as anti-tick vaccines against Haemaphysalis longicornis

BackgroundTick control is an essential aspect of controlling the spread of tick-borne diseases affecting humans and animals, but it presently faces several challenges. Development of an anti-tick vaccine is aimed at designing cost-effective and environmentally friendly protection against ticks and tick-borne diseases as an alternative to the use of chemical acaricides. A single vaccine from the tick midgut protein Bm86 is currently available for field applications, but its efficacy is limited to only some tick species. Identification of candidate vaccine antigens that can affect multiple tick species is highly desirable. The hard tick Haemaphysalis longicornis has two kinds of the iron-binding protein ferritin (HlFER), an intracellular HlFER1 and a secretory HlFER2, and RNA interference experiments showed that these are physiologically important in blood feeding and reproduction and in protection against oxidative stress. Here we investigated the potential of targeting HlFERs for tick control by immunizing the host with recombinant HlFERs (rHlFER1 and rHlFER2).MethodsRabbits were immunized with rHlFERs three times subcutaneously at two-week intervals. Antisera were collected before the first immunization and a week after each immunization to confirm the antigen-specific serum antibody titer by serum ELISA. Two weeks after the final immunization, the rabbits were challenged with tick infestation. After dropping, tick feeding and reproduction parameters were evaluated to determine vaccine efficacy. To demonstrate the effects of antibodies, oxidative stress was detected in the eggs and larvae.ResultsThe antibody titer of rHlFER-immunized rabbits greatly increased after the second immunization. Antibodies exhibited cross-reactivity with rHlFERs and reacted with tick native HlFERs in Western blot analysis. Significantly lower bodyweight was observed in the ticks infested from the rHlFER2-immunized rabbit compared to those from the control rabbit. Reduced oviposition and hatching rate were observed in both rHlFER-immunized groups. rHlFER2 showed a higher vaccine efficacy. The antibodies against rHlFERs were detected in the eggs, and higher levels of oxidative stress biomarkers in the eggs and larvae, of ticks from rHlFER vaccinated rabbits.ConclusionCollectively, these results showed that HlFER2 has a good potential as an anti-tick vaccine antigen that may affect multiple tick species.