Vector competence of Haemaphysalis longicornis ticks for a Japanese isolate of the Thogoto virus.

Thogoto virus (THOV), a tick-borne arbovirus not previously reported in East Asia, was recently isolated from Haemaphysalis longicornis in Kyoto, Japan. In this study, we investigated the vector competence of H. longicornis ticks for a Japanese isolate of the Thogoto virus using anal pore microinjection and experimental virus acquisition. Our results showed that anal pore microinjection can readily infect adult ticks, and THOV-infected ticks can successfully transmit the virus to mice. Blood feeding was also critical in the distribution of the virus in tick organs, most especially in the salivary glands. Furthermore, co-feeding between an infected adult and naïve nymphs can also produce infected molted adults that can horizontally transmit THOV to mice. Altogether, our results suggest that H. longicornis is a competent vector for the Japanese THOV isolate and could be the primary tick vector of the virus in Japan.

Immunofluorescent detection in the ovary of host antibodies against a secretory ferritin injected into female Haemaphysalis longicornis ticks.

Due to the continuous threat of ticks and tick-borne diseases to human and animal health worldwide, and the drawbacks of chemical acaricide application, many researchers are exploring vaccination as an alternative tick control method. Earlier studies have shown that host antibodies can circulate in the ticks, but it has not been confirmed whether these antibodies can be passed on to the eggs. We previously reported that ticks infesting rabbits immunized with a recombinant secretory ferritin of Haemaphysalis longicornis (HlFER2) had reduced egg production and hatching. Here we attempted to detect the presence of antibodies against HlFER2 in the ovary and eggs of female ticks through immunofluorescent visualization. Purified anti-HlFER2 antibodies or rabbit IgG for control was directly injected to engorged female H. longicornis. Ovaries and eggs after oviposition were collected and prepared for an indirect immunofluorescent antibody test. Positive fluorescence was detected in ovaries one day post-injection of anti-HlFER2 antibodies. Through silencing of Hlfer2 gene, we also determined whether the injected antibodies can specifically bind to native HlFER2. Immunofluorescence was observed in the oocytes of dsLuciferase control ticks injected with anti-HlFER2 antibodies, but not in the oocytes of Hlfer2-silenced ticks also injected with anti-HlFER2 antibodies. Our current findings suggest that host antibodies can be passed on to the oocytes, which is significant in formulating a vaccine that can disrupt tick reproduction.

Synchronous Langat Virus Infection of Haemaphysalis longicornis Using Anal Pore Microinjection.

The tick-borne encephalitis virus (TBEV) serocomplex of flaviviruses consists of arboviruses that cause important diseases in animals and humans. The transmission of this group of viruses is commonly associated with tick species such as Ixodes spp., Dermacentor spp., and Hyalomma spp. In the case of Haemaphysalis longicornis, the detection and isolation of flaviviruses have been previously reported. However, studies showing survival dynamics of any tick-borne flavivirus in H. longicornis are still lacking. In this study, an anal pore microinjection method was used to infect adult H. longicornis with Langat virus (LGTV), a naturally attenuated member of the TBEV serocomplex. LGTV detection in ticks was done by real-time PCR, virus isolation, and indirect immunofluorescent antibody test. The maximum viral titer was recorded at 28 days post-inoculation, and midgut cells were shown to be the primary replication site. The tick can also harbor the virus for at least 120 days and can successfully transmit LGTV to susceptible mice as confirmed by detection of LGTV antibodies. However, no transovarial transmission was observed from the egg and larval samples. Taken together, our results highly suggest that anal pore microinjection can be an effective method in infecting adult H. longicornis, which can greatly assist in our efforts to study tick and virus interactions.

Characterization and antiviral activity of a newly identified defensin-like peptide, HEdefensin, in the hard tick Haemaphysalis longicornis.

Tick defensins are antimicrobial peptides that play a major role in the innate immunity of ticks by providing a direct antimicrobial defense. In this study, we identified and characterized a defensin-like encoding gene, HEdefensin, from the expressed sequence tags (EST) database of hemolymph from the hard tick Haemaphysalis longicornis. Expression of the gene in whole adult ticks and in different organs was upregulated during blood feeding, though not after Langat virus (LGTV) challenge. A synthetic HEdefensin peptide demonstrated significant virucidal activity against LGTV but not against an adenovirus in co-incubation virucidal assays. Moreover, the RNAi-mediated gene silencing of HEdefensin did not significantly affect the virus titer as compared to the control group. The data reported here have established the in vitro virucidal activity of the peptide against LGTV. However, its role in the innate antiviral immunity of H. longicornis remains to be explored, and further studies are needed to fully evaluate the potential biological activities of the peptide against bacteria, fungi or parasites.

Establishment of a novel tick-Babesia experimental infection model.

Ticks are potent vectors of many deadly human and animal pathogens. Tick-borne babesiosis is a well-recognized malaria-like disease that occurs worldwide and recently has attracted increased attention as an emerging zoonosis. Although the proliferation of Babesia organisms is essential in the vectors, their detailed lifecycle with time information for migration in ticks remains unknown. A novel study model for the elucidation of the migration speed of Babesia parasites in their vector tick, Haemaphysalis longicornis, has been developed using an artificial feeding system with quantitative PCR method. The detectable DNA of Babesia parasites gradually disappeared in the tick midgut at 1 day post engorgement (DPE), and in contrary increased in other organs. The results indicated that the Babesia parasite passed the H. longicornis midgut within 24 hours post engorgement, migrated to the hemolymph, and then proliferated in the organs except the midgut. This time point may be an important curfew for Babesia parasites to migrate in the tick lumen. We also visualized the Babesia parasites in the experimentally infected ticks and in their eggs using IFAT for detecting their cytoskeletal structure, which suggested the successful tick infection and transovarial transmission of the parasite. This model will shed light on the further understanding of tick-Babesia interactions.

2-Cys peroxiredoxin is required in successful blood-feeding, reproduction, and antioxidant response in the hard tick Haemaphysalis longicornis.

BACKGROUND: Ticks are obligate hematophagous arthropods that feed on vertebrate blood that contains iron. Ticks also concentrate host blood with iron; this concentration of the blood leads to high levels of iron in ticks. The host-derived iron reacts with oxygen in the tick body and this may generate high levels of reactive oxygen species, including hydrogen peroxide (H2O2). High levels of H2O2 cause oxidative stress in organisms and therefore, antioxidant responses are necessary to regulate H2O2. Here, we focused on peroxiredoxin (Prx), an H2O2-scavenging enzyme in the hard tick Haemaphysalis longicornis. METHODS: The mRNA and protein expression profiles of 2-Cys peroxiredoxin (HlPrx2) in H. longicornis were investigated in whole ticks and internal organs, and developmental stages, using real-time PCR and Western blot analysis during blood-feeding. The localization of HlPrx2 proteins in tick tissues was also observed by immunostaining. Moreover, knockdown experiments of HlPrx2 were performed using RNA interference to evaluate its function in ticks. RESULTS: Real-time PCR showed that HlPrx2 gene expression in whole ticks and internal organs was significantly upregulated by blood-feeding. However, protein expression, except in the midgut, was constant throughout blood-feeding. Knockdown of the HlPrx2 gene caused significant differences in the engorged body weight, egg weight and hatching rate for larvae as compared to the control group. Finally, detection of H2O2 after knockdown of HlPrxs in ticks showed that the concentration of H2O2 significantly increased before and after blood-feeding. CONCLUSION: Therefore, HlPrx2 can be considered important for successful blood-feeding and reproduction through the regulation of H2O2 concentrations in ticks before and after blood-feeding. This study contributes to the search for a candidate target for tick control and further understanding of the tick's oxidative stress coping mechanism during blood-feeding.

Whole genome characterization of a G6P[5] rotavirus A strain isolated from a stray cat in Japan.

The whole genome of an unusual G6P[5] rotavirus A strain named FRV537, which was isolated from a stray cat in Japan, was characterized to determine its species of origin. The genotype constellation of FRV537 was G6-P[5]-I2-R2-C2-M2-A13-N2- T6-E2-H3. No known feline rotavirus has this genotype constellation; the Japanese equine strain OH-4 is the only known strain that does. While FRV537 shares the same genotype with some feline rotaviruses in all genes except those encoding VP4 and NSP1, none of these genes are closely related to those of known feline rotaviruses. By contrast, G6P[5] is almost exclusively present in bovine rotaviruses. The VP7 and VP4 genes of FRV537 formed a lineage with typical bovine rotaviruses with high bootstrap values. As to the internal capsid and nonstructural gene constellation, three bovine rotavirus strains had a constellation identical to that of FRV537. Moreover, each of the genotypes of FRV537 was found to be a common bovine genotype. In addition to the high nucleotide sequence identities between FRV537 and bovine rotaviruses in each genome segment (≥95%), phylogenetic analysis revealed a close relationship to bovine/artiodactyl rotaviruses. Thus, the molecular and phylogenetic evidence suggests that FRV537 isolated from a stray cat was of bovine rotavirus origin.

Host Immunization with Recombinant Proteins to Screen Antigens for Tick Control.

Ticks (Parasitiformes: Ixodida) are known for their obligate blood feeding habit and their role in transmitting pathogens to various vertebrate hosts. Tick control using chemical acaricides is extensively used particularly in livestock management, but several disadvantages arise from resistance development of many tick species, and concerns on animal product and environmental contamination. Vaccination offers better protection and more cost-effective alternative to application of chemical acaricides, addressing their disadvantages. However, an ideal anti-tick vaccine targeting multiple tick species and all the tick stages is still wanting. Here, we describe the procedures involved in the evaluation of a vaccine candidate antigen against ticks at the laboratory level, from the preparation of recombinant proteins, administration to the rabbit host and monitoring of antibody titer, to tick infestation challenge and determination of the effects of immunization to ticks.

Induction of gene silencing in Haemaphysalis longicornis ticks through immersion in double-stranded RNA.

The continuous emergence of tick-borne diseases and chemical acaricide-resistant tick strains necessitates the development of new and more effective control strategies. RNA interference through the injection of double-stranded RNA (dsRNA) has been a very useful tool in tick research for evaluating gene function. However, this technique can be sophisticated due to the required equipment and technique. Here we studied the feasibility of an immersion technique to induce gene silencing in Haemaphysalis longicornis ticks. We targeted the Hlfer1 gene, previously shown to be crucial in successful blood feeding and reproduction. Larval, nymphal, and adult female H. longicornis ticks were immersed in Hlfer1 or Luciferase dsRNA for control. The dsRNA dissolving medium, incubation temperature and time were varied to establish the optimum conditions. RT-PCR was performed to confirm gene silencing. It was found that immersing the ticks in dsRNA dissolved in nuclease-free water at 15°C for 12h resulted in clear gene silencing. The phenotypes of adult ticks immersed in dsRNA were then compared with those of adult ticks injected with dsRNA. Similar to dsRNA injection, the post-blood meal weight of ticks immersed in Hlfer1 dsRNA was significantly lower than the control group. Moreover, high post-blood meal mortality and low egg output was observed both from ticks injected with and immersed in Hlfer1 dsRNA. Our results here suggest that immersion in dsRNA can effectively induce gene silencing and not only offers an alternative method to dsRNA injection but also opens the possibility of applying dsRNA for tick control.

Improvement of the cryopreservation method for the Babesia gibsoni parasite by using commercial freezing media.

In vitro cultivation and cryopreservation under liquid nitrogen have already been reported and established for Babesia bovis and Babesia bigemina parasites. Although the in vitro cultivation methods for Babesia gibsoni have been reported and established, the cryopreservation methods for this parasite have not been established completely. In this paper, we compared several freezing media for the cryopreservation of B. gibsoni parasite. The CELLBANKER® series (1 plus and 2), STEM-CELLBANKER®, and CultureSure® were used for commercial freezing media; 10% dimethyl sulfoxide in 90% fetal bovine serum, 20% polyvinylpyrrolidone in phosphate-buffered saline (established for bovine Babesia parasites), and 28% glycerol supplemented with 3% sorbitol and 0.65% NaCl dissolved in water (established for Plasmodium parasites) were used for conventional media. Our results demonstrated that the CELLBANKER® series (1 plus and 2), STEM-CELLBANKER®, and CultureSure® are effective freezing media for B. gibsoni parasite compared to the cryopreservation methods of bovine Babesia and Plasmodium parasites. Our improved method of cryopreservation would contribute to the stability of the in vitro cultivation of B. gibsoni parasite.

Virucidal activity of Haemaphysalis longicornis longicin P4 peptide against tick-borne encephalitis virus surrogate Langat virus.

BACKGROUND: Longicin is a defensin-like peptide, identified from the midgut epithelium of hard tick Haemaphysalis longicornis. Several studies have already shown the antimicrobial and parasiticidal activities of longicin peptide and one of its synthetic partial analogs, longicin P4. In this study, longicin peptides were tested for potential antiviral activity against Langat virus (LGTV), a tick-borne flavivirus. METHODS: Longicin P1 and P4 peptides were chemically synthesized. Antiviral activity of the longicin peptides against LGTV was evaluated through in vitro virucidal assays, wherein the antiviral efficacy was determined by reduction in number of viral foci and virus yield. Additionally, longicin P4 was also tested for its activity against human adenovirus, a non-enveloped virus. Lastly, to assess the importance of longicin on the innate antiviral immunity of H. longicornis ticks, gene silencing through RNAi was performed. RESULTS: Longicin P4 produced significant viral foci reduction and lower virus yield against LGTV, while longicin P1 failed to demonstrate the same results. Conversely, both longicin partial analogs (P1 and P4) did not show significant antiviral activity when tested on adenovirus. In addition, longicin-silenced ticks showed significantly higher virus titer after 7 days post-infection but a significantly lower titer was detected after an additional 14 days of observation as compared to the Luc dsRNA-injected ticks. Mortality in both groups did not show any significant difference. CONCLUSION: Our results suggest that longicin P4 has in vitro antiviral activity against LGTV but not against a non-enveloped virus such as adenovirus. Likewise, though most cationic antimicrobial peptides like longicin act directly on target membranes, the exact mechanism of membrane targeting of longicin P4 in enveloped viruses, such as LGTV, requires further investigation. Lastly, while the in vitro virucidal capacity of longicin P4 was confirmed in this study, the role of the endogenous tick longicin in the antiviral defense of H. longicornis against LGTV still remains to be demonstrated.

Impaired cellular immune response to injected bacteria after knockdown of ferritin genes in the hard tick Haemaphysalis longicornis.

Iron is an indispensable element for most microorganisms, including many pathogenic bacteria. Iron-withholding is a known component of the innate immunity, particularly of vertebrate hosts. Ticks are vectors of multiple pathogens and reports have shown that they naturally harbor several bacterial species. Thus, tick innate immunity must be crucial in limiting bacterial population to tolerable level that will not cause adverse effects. We have previously characterized two types of the iron-binding protein ferritin (HlFER) in the hard tick Haemaphysalis longicornis, known to be a vector of some protozoan parasites and rickettsiae, and showed their antioxidant function and importance in blood feeding and reproduction. Here we examined the possible role of HlFERs in tick immunity against bacterial infection. After silencing Hlfer genes, adult ticks were injected with live enhanced green fluorescence protein-expressing Escherichia coli, and then monitored for survival rate. Hemolymph that included hemocytes was collected for microscopic examination to observe cellular immune response, and for E. coli culture to determine bacterial viability after injection in the ticks. The expression of some antimicrobial peptides in whole ticks was also analyzed by RT-PCR. Hlfer-silenced ticks had a significantly lower survival rate than control ticks after E. coli injection. Greater number of bacteria inside and outside the hemocytes and higher bacterial colony counts after culture with hemolymph were also observed in Hlfer-silenced ticks. However, no difference on the expression of antimicrobial peptides was observed. These results suggest that ferritin molecules might be important in the cellular immune response of ticks to some bacteria.

A novel C-type lectin with triple carbohydrate recognition domains has critical roles for the hard tick Haemaphysalis longicornis against Gram-negative bacteria.

C-type lectins (CLecs) play an important role in innate immunity against invaders. In this study, a novel CLec was identified from Haemaphysalis longicornis ticks (HlCLec). HlCLec contains a signal peptide and a transmembrane region. Interestingly, HlCLec possesses three dissimilar carbohydrate recognition domains (CRDs). Each CRD contains the mutated motif of Ca(2+)-binding site 2. HlCLec mRNA was up-regulated during blood feeding, and had highest expression in the midgut and ovary. HlCLec localization was also confirmed by immunofluorescent antibody test (IFAT). HlCLec was found on the cell membrane and basal lamina of midgut and ovary. In addition, the recombinant HlCLec and individual CRDs demonstrated direct binding activity to Escherichia coli and Staphylococcus aureus; however, no growth inhibition activity was observed. Furthermore, E. coli injection after silencing of HlCLec caused drastic reduction in survival rate of ticks. These results strongly suggest the key role of HlCLec in tick innate immunity against Gram-negative bacteria.

Role of the tumor necrosis factor receptor-associated factor-type zinc finger domain containing protein 1 (TRAFD1) from the hard tick Haemaphysalis longicornis in immunity against bacterial infection.

A tumor necrosis factor receptor-associated factor-type zinc finger domain containing protein 1 (TRAFD1) is a negative feedback regulator that controls excessive immune responses in vertebrates. The sequence of tick hemolymph TRAFD1 from the hard tick Haemaphysalis longicornis (HlTRAFD1) was analyzed after identification and cloning from the expressed sequence tag database. RT-PCR and Western blot analyses showed that HlTRAFD1 transcript and protein levels after blood feeding were present in all developmental stages, and the transcript level was consistently high in all organs examined from adult female ticks upon engorgement. Knockdown of HlTRAFD1 gene by RNA interference did not affect blood feeding or oviposition. However, HlTRAFD1 silencing affected the expression of the longicin gene, a defensin-like molecule, but not the lysozyme gene. Moreover, the survival rate of HlTRAFD1-silenced ticks was lower, and the number of E. coli was higher in the hemolymph and plasmatocytes after E. coli injection compared to the control group. These results suggested that HlTRAFD1 strongly affected both the humoral and cellular immunity of ticks.

The widely distributed hard tick, Haemaphysalis longicornis, can retain canine parvovirus, but not be infected in laboratory condition.

Ticks are known to transmit various pathogens, radically threatening humans and animals. Despite the close contact between ticks and viruses, our understanding on their interaction and biology is still lacking. The aim of this study was to experimentally assess the interaction between canine parvovirus (CPV) and a widely distributed hard tick, Haemaphysalis longicornis, in laboratory condition. After inoculation of CPV into the hemocoel of the ticks, polymerase chain reaction assay revealed that CPV persisted in inoculated unfed adult female ticks for 28 days. Canine parvovirus was recovered from the inoculated ticks using a cell culture, indicating that the virus retained intact in the ticks after inoculation, but significant positive reaction indicating virus infection was not detected in the tick organs by immunofluorescence antibody test using a monoclonal antibody. In the case of ticks inoculated with feline leukemia virus, the virus had shorter persistence in the ticks compared to CPV. These findings provide significant important information on the characteristic interaction of tick with non-tick-borne virus.

Identification of the Babesia-responsive leucine-rich repeat domain-containing protein from the hard tick Haemaphysalis longicornis.

Haemaphysalis longicornis is a tick known for transmitting Babesia parasites, including Babesia gibsoni, in East Asian countries. The vector tick must have strategies to control Babesia parasites, while Babesia parasites are also considered to establish an evasive mechanism from the tick's innate immunity. Due to this mutual tolerance, H. longicornis is considered to be a vector of Babesia parasites. Recent studies have shown the important roles of leucine-rich repeat (LRR) domain-containing proteins in innate immunity in many living organisms. Some LRR domain-containing proteins were identified in ticks; however, their functions are still unknown. In this study, a novel LRR domain-containing protein was identified from H. longicornis (HlLRR). HlLRR contains two LRR domains, and the expression levels of mRNA and proteins were upregulated during blood feeding, particularly in the salivary glands and midgut. In addition, recombinant HlLRR (rHlLRR) demonstrated growth inhibition activity against B. gibsoni in vitro without a hemolytic effect at any concentration used. Moreover, the diameters of Babesia merozoites treated with rHlLRR were significantly larger than those of the control group. These results strongly indicate the key roles of HlLRR in the tick's innate immunity against Babesia parasites. Furthermore, HlLRR might be a potential alternative drug to treat babesiosis.

Whole genotype constellation of prototype feline rotavirus strains FRV-1 and FRV64 and their phylogenetic relationships with feline-like human rotavirus strains.

Feline rotaviruses, members of the species Rotavirus A, are an infrequent source of zoonotic infections, and were previously shown by RNA-RNA hybridization assays to possess two distinct genomic RNA constellations, represented by strains FRV-1 and FRV64. Due to the lack of whole genome sequence information for FRV-1, human rotavirus strain AU-1 has been used as a surrogate for the genotype constellation of feline rotaviruses. The aim of this study was to determine the whole genome sequence of FRV-1 and FRV64 to help understand the genetic relationships among existing feline rotaviruses from the evolutionary perspective. The genotype constellations of FRV-1 and FRV64 were G3-P[9]-I3-R3-C3-M3-A3-N3-T3-E3-H3 and G3-P[3]-I3-R3-C2-M3-A9-N2-T3-E3-H6, respectively. FRV-1 has a genotype constellation identical to that of the AU-1 strain. Although for individual genes they shared lineages, with the exception of genes encoding VP2, VP6 and VP7, the sequence identity between FRV-1 and AU-1 was considered to be sufficiently high for the AU-1 to be regarded as an example of the direct transmission of a feline rotavirus to a child. On the other hand, the FRV64 strain was not only similar in all the 11 genome segments to another feline rotavirus strain, Cat97, but also to canine rotavirus strains (K9 and CU-1) and feline/canine-like human rotavirus strains (Ro1845 and HCR3A). In conclusion, this study revealed intermingled sharing of genotypes and lineages among feline rotaviruses, suggesting the occurrence of frequent reassortment events over the course of evolution to emerge in four genotype constellations represented by FRV-1, FRV64/Cat97, Cat2 and BA222 strains.

Detection of Norovirus and Sapovirus from diarrheic dogs and cats in Japan.

Norovirus (NoV) and sapovirus (SaV) are important causes of human diarrhea. In this study, between 2007 and 2014 fecal samples were collected from 97 dogs and 83 cats with diarrhea and examined to determine the prevalence of NoV and SaV infections in Japan. To detect caliciviruses, approximately 300 bases targeting the polymerase gene were amplified using RT-PCR and subjected to phylogenetic and homology analyses. Specific PCR products were obtained from four canine and nine feline samples: two canine and one feline isolate were classified as NoV, two canine isolates as SaV and the remaining eight feline isolates as vesivirus (VeV). The three NoV isolates were classified into the same clade as that of known canine and feline NoVs; their homologies (75.9-92.3%) were higher than those with human genogroup IV (GIV) NoVs (59.1-65.9%). The homology of the feline NoV isolate with previously reported feline NoV isolates was particularly high (91.7-92.3%). Regarding SaV, the two canine isolates were classified into the same clade as known canine SaVs and their homologies (72.5-86.5%) were higher than those with other mammal SaVs (20.7-58.0%). The eight feline VeV isolates were assumed to be feline calicivirus. The present study is the first report of the presence of NoV- and SaV-infected dogs and cats in Japan. The findings suggest there are species-specific circulations of NoV and SaV among dogs and cats, in Japan.

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.

The haemagglutination activity of equine herpesvirus type 1 glycoprotein C.

Equine herpesvirus type 1 (EHV-1) has haemagglutination (HA) activity toward equine red blood cells (RBCs), but the identity of its haemagglutinin is unknown. To identify the haemagglutinin of EHV-1, the major glycoproteins of EHV-1 were expressed in 293T cells, and the cells or cell lysates were mixed with equine RBCs. The results showed that only EHV-1 glycoprotein C (gC)-producing cells adsorbed equine RBCs, and that the lysate of EHV-1 gC-expressing cells agglutinated equine RBCs. EHV-1 lacking gC did not show HA activity. HA activity was inhibited by monoclonal antibodies (MAbs) specific for gC, but not by antibodies directed against other glycoproteins. In addition, HA activity was not inhibited by the addition of heparin. These results indicate that EHV-1 gC can bind equine RBCs irrespective of heparin, in contrast to other herpesvirus gC proteins.