Prevalence of honeybee viruses in different regions of China and Argentina.

Honeybees are threatened by various pathogens and parasites. More than 18 viruses have been described in honeybees and many of them have been detected in China and Argentina. In China, both Apis cerana and Apis mellifera are raised. In Argentina, beekeepers raise different ecotypes of A. mellifera: European honeybees (in both temperate and subtropical regions) and Africanised honeybees (in subtropical areas only). A thorough study was carried out in both China and Argentina to analyse the current virus presence and distribution in different climatic zones and gather information on different bee species/subspecies. Adult honeybees were collected from apiaries in temperate and subtropical regions of China (including areas with exclusive populations of A. mellifera, areas where A. mellifera and A. cerana co-exist, and areas with exclusive populations of A. cerana) and Argentina. Six viruses, namely, deformed wing virus (DWV), black queen cell virus (BQCV), sacbrood virus (SBV), chronic bee paralysis virus (CBPV), acute bee paralysis virus (ABPV) and Israeli acute paralysis virus (IAPV) were detected in China, both in A. cerana and in A. mellifera, while four viruses (DWV, BQCV, CBPV and ABPV) were present in Argentina. Interestingly, multiple infections were commonly found in China, with up to five different viruses co-circulating in some colonies without apparent abnormalities. In this study, no Chinese samples were positive for slow bee paralysis virus. The most prevalent viruses were BQCV (China) and DWV (Argentina). Kashmir bee virus was absent from samples analysed for both countries.

Les populations d'abeilles mellifères sont menacées par de nombreux agents pathogènes et parasites. Parmi eux, 18 virus ont été décrits, dont plusieurs ont été détectés en Chine et en Argentine. Les espèces d'abeilles mellifères élevées en Chine sont Apis cerana et Apis mellifera. En Argentine, les apiculteurs élèvent plusieurs écotypes d'A. mellifera : le type européen dans les régions tempérées et subtropicales et le type africanisé dans les zones subtropicales. Une étude approfondie a été réalisée en Chine et en Argentine dans le but d'identifier les virus présents, d'analyser leur distribution dans différentes zones climatiques et de réunir des informations sur les différentes espèces et sous-espèces d'abeilles présentes. Des abeilles mellifères adultes ont été collectées dans des ruchers des régions tempérées et subtropicales de Chine (zones peuplées exclusivement d'A. mellifera ou d'A. cerana et zones où A. mellifera et A. cerana coexistent) et d'Argentine (A. mellifera seulement). En Chine, six virus, à savoir le virus des ailes déformées, le virus des cellules royales noires, le virus du couvain sacciforme, le virus de la paralysie chronique de l'abeille, le virus de la paralysie aiguë de l'abeille et le virus israélien de la paralysie aiguë ont été détectés aussi bien chez A. cerana que chez A. mellifera ; en Argentine, quatre virus ont été détectés (virus des ailes déformées, virus des cellules royales noires, virus de la paralysie chronique de l'abeille et virus de la paralysie aiguë de l'abeille). Fait intéressant, les infections multiples étaient fréquentes en Chine, avec parfois jusqu'à cinq virus différents circulant dans certaines colonies sans provoquer de manifestations anormales apparentes. Aucun des échantillons analysés en Chine n'a été trouvé positif pour le virus de la paralysie lente de l'abeille. Les virus les plus fréquents étaient, en Chine, le virus des cellules royales noires et en Argentine, le virus des ailes déformées. Le virus du Cachemire n'a été trouvé dans aucun des échantillons analysés dans les deux pays.

Las abejas melíferas están amenazadas por diversos patógenos y parásitos. Se han descrito más de 18 virus que las afectan, muchos de los cuales se han detectado en China y la Argentina. En China se cultivan tanto Apis cerana como Apis mellifera, mientras que los apicultores argentinos crían diferentes ecotipos de A. mellifera: abejas europeas en las regiones templadas y subtropicales y abejas africanizadas en las zonas subtropicales. Los autores exponen un minucioso estudio realizado a la vez en China y la Argentina con el fin de analizar la actual presencia y distribución de virus en diferentes zonas climáticas y reunir información sobre distintas especies y subespecies de abeja. En primer lugar se recogieron abejas adultas de colmenares situados en regiones templadas y subtropicales de China (algunas donde hay exclusivamente poblaciones de A. mellifera, otras donde coexisten A. mellifera y A. cerana y otras zonas que albergan solo poblaciones de A. cerana) y la Argentina (solamente A. mellifera). En las poblaciones chinas tanto de A. cerana como de A. mellifera se detectaron seis virus: virus de las alas deformes (VAD); virus de las celdas reales negras (VCRN); virus de la cría ensacada (VCE); virus de la parálisis crónica de la abeja (VPCA); virus de la parálisis aguda de la abeja (VPAA); y virus de la variante israelí del virus de la parálisis aguda (VPAI), mientras que en la Argentina se observó la presencia de cuatro virus (VAD, VCRN, VPCA y VPAA). Un dato interesante es que en China se observaron con frecuencia infecciones múltiples, con hasta cinco virus diferentes circulando a la vez en algunas colonias sin que ello diera lugar a anormalidades aparentes. Ninguna de las muestras chinas analizadas en el estudio resultó positiva al virus de la parálisis lenta de la abeja. Los virus más prevalentes fueron el VCRN (China) y el VAD (Argentina). El virus Cachemira de las abejas estaba ausente de las muestras analizadas en ambos países.

Early protection against foot-and-mouth disease virus in cattle using an inactivated vaccine formulated with Montanide ESSAI IMS D 12802 VG PR adjuvant.

Foot and mouth disease is an acute disease of cattle with a broad distribution around the world. Due to the fast spread of FMDV infections, control measures must be applied immediately after an outbreak, such as the use of vaccines that induce fast protection. Previously, it was shown that mice vaccinated with FMD inactivated virus (iFMDV) formulated with Montanide™ ESSAI IMS D 12802 VG PR adjuvant (802-iFMDV) were protected when they were challenged 4 and 7 days post-vaccination (dpv) with homologous virus. In this work, we describe the successful use of this formulation in cattle. In addition, adjuvant Montanide™ IMS 1313 VG NPR was also tested. 802-iFMDV vaccine was able to confer 100% protection against viral challenge at 4 and 7 dpv, while eliciting low antibody levels, at 7 dpv. 1313-iFMDV vaccine induced protection in 60% of cattle. At 4 dpv, 1313-iFMDV vaccinated animals presented increased levels of IFNγ but not of macrophages. At 4 and 7 dpv, macrophages, IFNγ, nasal IgA and IgG1 antibodies against FMDV, and opsonophagocytosis were increased in animals vaccinated with 802-iFMDV indicating that these phenomena could be involved in protection.It is the first time that total protection against FMDV at early stages post-vaccination is reported using a single dose of the formulation iFMDV plus Montanide™ ESSAI D IMS 12802 VG PR adjuvant.

Validation of an r3AB1-FMDV-NSP ELISA to distinguish between cattle infected and vaccinated with foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed livestock which has a drastic economic impact for affected countries. Although FMDV is distributed worldwide, many regional programs have been effective eradicating this agent. In Argentina, as in many other regions of South America, the combination of a systematic vaccination plan, together with an effective detection system capable of differentiating infection from vaccination, has been successful for eradicating this agent from the country. The properties of recombinant 3AB1 FMDV non-structural protein (r3AB1 FMDV-NSP), as a marker for the detection of antibodies to differentiate between cattle infected and vaccinated with FMDV, have been described previously. The goal of the present study was to validate the 3AB1 ELISA using a well characterized serum panel from Argentina (n=559) including eight national and one international reference sera. Overall, the 3AB1 ELISA demonstrated good feasibility, repeatability, reproducibility, analytical sensitivity and specificity, and accuracy. The results from the 3AB1 ELISA when compared with those obtained from the OIE index test (NCPanaftosa screening) showed a similar performance of both tests [diagnostic sensitivity=84% (C.I.=79-88%) and 80% (C.I.=75-85%), respectively; and diagnostic specificity=98.6% (C.I.=97-100%) and 95% (C.I.=91-98%), respectively]. The present work proposes the 3AB1 ELISA as an alternative to imported kits for FMD internal screening and transboundary sero-surveillance.

Detection of bovine leukemia virus specific antibodies using recombinant p24-ELISA.

We developed an indirect ELISA test (rp24-ELISA) for the detection of antibodies against the p24 capsid protein of bovine leukemia virus (BLV) in bovine serum samples. A recombinant p24 (rp24) was expressed in the Escherichia coli expression system, purified in a nickel charged resin and used as antigen in the ELISA test. A cut-off point was established considering the distribution of reactivity values obtained by rp24-ELISA on a set of 555 field serum samples that were stated as double positive (DP) or double negative (DN) by the combination of commercial agar gel immunodiffusion (AGID) assay and gp51-ELISA tests results. The rp24-ELISA showed good concordance with the agar gel immunodiffusion assay, when 710 serum samples were analyzed. In addition, rp24-ELISA demonstrated to be a precise assay with good repeatability and reproducibility and a better analytical sensitivity than AGID. From 67 discordant rp24-ELISA-AGID sera, 4 negative reactors were from the DP group, 28 positive samples were from the DN group and 35 positive samples were stated as negative by AGID but positive by the gp51-ELISA. Samples from this last subgroup were sent to an OIE reference laboratory where 28 samples were stated as negative, 5 samples were stated as positive and 2 as inconclusive. Complete concordance with blind previous results from an international proficiency test panel confirmed the capability of the assay to discriminate between infected and non-infected animals. In conclusion, the rp24-ELISA developed and standardized demonstrated to have good analytical characteristics to be considered for screening of BLV.

Foot-and-mouth disease vaccine potency testing in cattle using homologous and heterologous challenge strains: precision of the "Protection against Podal Generalisation" test.

The level of protection conferred by foot-and-mouth disease (FMD) vaccines in primovaccinated animals primarily depends on the potency of the vaccine and the relatedness of the vaccine strain and circulating field isolate. The "Gold Standard" FMD vaccine potency test is the in vivo test performed in the target species. The objective of the study was to determine the precision of the in vivo "Protection against Podal Generalisation" (PPG) FMD vaccine potency test in cattle using homologous (vaccine quality control) and heterologous (vaccine matching) viral challenge. The overall level of protection induced by the A(24) Cruzeiro/Brazil/55 vaccine used in six homologous PPG tests was 88.5%. Vaccine accordance (VACC) and vaccine concordance (VCON) were estimated to be 75.9% and 73.7%, respectively. In four heterologous challenge PPG tests, the overall level of cross-protection induced by the A(24) Cruzeiro/Brazil/55 vaccine against A Argentina/2001 challenge was 26.6%, with VACC and VCON values of 65.7% and 59.2%, respectively. Results indicate that the homologous PPG test is more reliable than the European Pharmacopoeia potency test, but that a larger number of animals should be used in order to increase the test's statistical power. In this regard, indirect alternative tests for vaccine potency and vaccine matching merit consideration.

Use of new adjuvants in an emergency vaccine against foot-and-mouth disease virus: evaluation of conferred immunity.

The ability of an emergency adjuvanted FMDV vaccine to elicit early protective immune response in mice was examined. We studied the efficacy of several adjuvants to induce such protection. The aqueous IMS1313 plus inactivated FMDV induce a higher protective immune response than the vaccine with inactivated virus alone at seven days post vaccination (dpv). Mice vaccinated with this formula showed higher lymphoproliferative index values and higher IL-2, IL-4 and IFNgamma levels than the controls.

Development of a multiplex polymerase chain reaction for the differentiation of bovine herpesvirus-1 and -5.

Bovine herpesvirus-1 (BHV-1) and bovine herpesvirus-5 (BHV-5) are closely related viruses which exhibit some important differences at the genetic and immunogenic levels which may explain the differences in their pathogenicity and epidemiological characteristics. A multiplex polymerase chain reaction (M-PCR) was developed to detect and differentiate between BHV-1 and BHV-5. In this M-PCR two pairs of primers (TK1, TK2 and GD1, GD2) were used in the same reaction mix to amplify a thymidine kinase genomic region (183 bp) of BHV-1 and one genomic region of the gLycoprotein D (564 bp) of BHV-5. The specificity of the M-PCR was demonstrated when using both primers pairs simultaneously with BHV-1 and BHV-5 templates. The two expected bands were amplified without the apparition of non-specific products. However, when other herpesvirus strains were used, there was no amplification. To evaluate the sensitivity of the assay, dilutions of purified viral DNA were made for M-PCR amplification. The detection limit was 7 pg for BHV-1 and 22 pg for BHV-5. It was also determined by comparing the M-PCR with viral isolation. M-PCR was able to detect one log10 more than viral isolation for BHV-1 and for BHV-5 was two logarithms lower. The applicability of M-PCR was demonstrated on different specimens. Twenty isolates from field samples (11 BHV-1 and nine BHV-5) were positive by M-PCR, and the results were completely coincident with previous characterization using the immunoperoxidase assay. M-PCR could detect viral DNA in organ samples from natural infections, such as semen and brain. In addition, M-PCR detected more positive samples than observation of the citophatic effect in cell culture of nasal swabs from experimentally infected animals in two different assays. Owing to the difference in size of the M-PCR products which allows easy identification in an electrophoretic run, it is not necessary to use extra blotting and hybridization steps or a second round of amplification to differentiate clearly between BHV-1 and BHV-5.

Serological survey of viral antibodies in llamas (Lama glama) in Argentina.

This study analysed sera from 390 llamas (Lama glama) from nine farms located in three different Argentine provinces: Buenos Aires, Cordoba and Jujuy. The samples were tested for antibodies against 8 virus known to infect cattle: bovine herpesvirus type 1 (BHV-1), bovine viral diarrhea virus (BVDV), bovine adenovirus (BAdV III), bovine enterovirus (BEV), bovine rotavirus (BRV), bluetongue virus (BTV), bovine leukaemia virus (BLV), and foot-and-mouth virus (FMDV) by conventional methods such as seroneutralization, immunoperoxidase staining, and agar gel immunodiffusion. The antibody prevalences detected in llamas were: BHV-1 in 0.77% (3/390), BVDV in 2.05% (8/390), BAdV III in 5.13% (20/390), BEV in 4.10% (16/390), BRV in 87.69% (342/390). No antibodies against BTV, BLV and VIAA (FMDV infection associated antigen) were detected.

Assessment using ELISA of the herd immunity levels induced in cattle by foot-and-mouth disease oil vaccines.

The development of a liquid-phase blocking sandwich ELISA (LPBE) to measure antibodies (Ab) produced in cattle with the O, A and C foot-and-mouth disease virus (FMDV) types of commercial vaccines used in Argentina is described. The test was specific: 99% of naïve cattle sera (n = 130) gave titres below log10 = 1.2, and none had a titre above log10 = 1.5. Comparative studies with serum neutralization test (SNT) using sera from cattle which received one or more vaccine doses is reported. The overall rank correlation coefficient (Spearman's rho, rs) between SNT and LPBE were highly significant (rs > 0.67, P < 0.0001) for all vaccine strains. LBPE Ab titres on sera collected 90 days post vaccination were compared with results of cattle protection tests by applying a logistic regression. The minimum Ab titres at which 85% and 75% of the cattle were protected for each FMDV type were determined in order to interpret field Ab data in terms of protection. Application of this method allows large scale serological examinations to monitor antibody levels in vaccinated animals as an indirect indicator of the FMD control program status in the field. Its use in the evaluation of commercial batches of FMD vaccine is discussed.

[Seroepidemiologic indicators for the evaluation of campaigns for the control of foot-and-mouth disease]. / Indicadores seroepidemiológicos para la evaluación de las campañas de control de fiebre aftosa.

A sero-epidemiological survey was conducted in two districts in Argentina between 1993 and 1995, to provide additional information on the epidemiology of foot and mouth disease (FMD) in Argentina and to assess the level of immunity in cattle populations, and the circulation of FMD virus. As part of the final stage of this survey, a comparison was made of the results obtained by the enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion techniques. Levels of population immunity against the four types of virus included in the vaccine increased progressively during the period of the survey until, in 1995, at the end of the vaccination period, the percentage of animals possessing adequate levels of protection was approximately 77% in yearlings, and more than 94% in cattle over one year old. During the three-year study, there was a clear tendency for viral activity to diminish, until in 1995 when between 3% and 0.6% were positive to the agar gel immunodiffusion test for the antigen associated with viral infection. By contrast, the ELISA detected antibody in about five times as many animals. The authors show how the increase in the level of population immunity was accompanied by a fall in viral activity.

Detection of virus infection-associated antigen and 3D antibodies in cattle vaccinated against foot and mouth disease.

The analysis of sera obtained from animals vaccinated or revaccinated with inactivated vaccines against foot and mouth disease (FMD) virus showed that these vaccines induced antibodies against the virus infection-associated (VIA) antigen, detectable by agar gel immunodiffusion (AGID). The present study evaluates the antibody response to protein 3D and the VIA antigen (VIAA) of FMD virus induced by different vaccines in a group of 51 calves. This response was detected using AGID and a liquid-phase blocking sandwich enzyme-linked immunosorbent assay (ELISA) for anti-3D antibodies (ELISA-3D). No anti-VIAA or anti-3D antibodies were detected after the initial vaccination. Following revaccination, animals giving positive results were detected by both methods. This immune response disappeared 60-120 days post-revaccination (dprv) according to the AGID method, and 90-180 dprv when ELISA-3D was used. Samples of oesophageal-pharyngeal fluid obtained from animals that remained positive for anti-VIAA antibodies at 90-120 dprv gave negative results for viral isolation, indicating that the transitional antibody response induced by the vaccine was due to the presence of non-structural antigens in the vaccine and not to viral infection. These results indicate that the ELISA-3D method could be used as a complementary method for sero-epidemiological studies as an indirect indicator of viral activity, as long as the age and vaccination status of the animals being sampled are taken into consideration.

Detection of antibodies against foot-and-mouth disease virus using a liquid-phase blocking sandwich ELISA (LPBE) with a bioengineered 3D protein.

A liquid-phase blocking sandwich enzyme-linked immunosorbent assay (ELISA-3D) was developed to detect specific antibodies to the 3D protein in sera from foot-and-mouth disease (FMD) virus (FMDV)-infected animals. The assay uses a nonstructural 3D recombinant protein and two polyclonal antisera, one for capture (bovine) and the other for detector (guinea pig). The specificity of the assay was demonstrated by negative results with 101 sera of cattle from the FMD-free zone in Argentina and with bovine and porcine sera raised against various RNA and DNA viruses. The ELISA-3D was able to detect antibodies in cattle after natural or experimental infection with FMDV of A, O, or C types as early as 5 days postinfection and at later stages in persistently infected animals. Comparison of the results with those obtained with the routinely used agar gel immunodiffusion test and a previously described ELISA, both employing a partially purified virus-infection-associated antigen, shows that the ELISA-3D is highly sensitive and specific and gives reproducible results. Its use as a tool for monitoring viral activity and for certification of FMDV-free animals is recommended.

Susceptibility of llamas (Lama glama) to infection with foot-and-mouth-disease virus.

An experimental trial was conducted to evaluate the ability of foot-and-mouth-disease (FMD) virus (serotypes A79, C3, O1) to infect susceptible llamas exposed either directly to affected livestock, or indirectly to llamas that had been directly exposed to affected livestock. In addition, susceptible livestock species (cattle, pigs, goats, and sheep) were exposed to those llamas that had been both directly and indirectly exposed to the FMD virus to further look at potential transmission possibilities. Of 30 llamas directly exposed to the FMD virus, only three (3/30) showed evidence of infection, and of those, only two (2/30) had mild clinical signs. No FMD virus was isolated from either oesophageal-pharyngeal (OP) fluid or blood samples collected from the infected llamas beyond 14 days post-exposure. There was no evidence of virus transmission between the directly exposed and indirectly exposed llamas or between both groups of llamas and susceptible domestic livestock, as determined by the lack of clinical signs, by virus isolation, and by serology results. These results provide further evidence that llamas are resistant to FMD infection, and that they play a minor role, if any, in transmitting the virus to domestic livestock.

Immune response of calves to foot-and-mouth disease virus vaccine emulsified with oil adjuvant. Strategies of vaccination.

Calves born to vaccinated cows under the regular annual vaccination programme were vaccinated at different ages using commercial quadrivalent (01, A79, A87 and C85 FMDV strains) vaccine emulsified in oil adjuvant. The antibody responses of vaccinated calves were evaluated using liquid-phase blocking sandwich ELISA. All calves 20, 30 and 40 days old having high maternal antibody titres responded well to vaccination. Moreover, 25-57% of vaccinated calves showed protective antibody titres both at 90 and 120 days post-vaccination (d.p.v.), whereas none of the non-vaccinated animals achieved these levels. Calves aged 3-4 months with non-protective levels of colostral-derived antibodies responded with high antibody titres to vaccination which persisted for at least 4 months. In both groups of calves a certain degree of suppression of postvaccinal response was observed which was related to colostral antibody titres. Our results suggest that in order to reduce the proportion of calves susceptible to infection it is advisable to immunize calves as young as 20 days old to induce acceptable antibody titres for the following 4 months. In addition, a second vaccination 60 d.p.v. ensures high antibody levels in high disease risk areas.

Foot-and-mouth disease virus (FMDV) experimental infection: susceptibility and immune response of adult mice.

Adult mice are susceptible to foot-and-mouth disease virus (FMDV) infection only under some experimental conditions. This paper report the results of pathogenesis studies on 4 different strains of mice (CF1, C3H, NIH-nude, BALB-c/J) infected with the cloned and uncloned 0(1)C strain of FMDV. High virus titers were detected in blood and pancreas 12-24 h after infection (p.i.); these persisted for up to 48 h p.i. in CF1 and BALB-c/J mice and 72 h p.i. in the two other mouse strains. Virus titers observed in other organs were lower than those found in blood. In pancreas, and occasionally in salivary glands, oropharynx, heart and testicles, viral antigen was detected by direct immunofluorescent assay. Circulating neutralizing antibodies appeared in CF1 and C3H mice at 72 and 96 h p.i. respectively, and their titers remained unchanged during the 30-day experimental period. Antibodies against viral infection-associated antigen (VIA) were detected for a shorter period. In animals irradiated with 1 LD 50 (total body irradiation), viremia persisted up to 14 days p.i. and a low antibody response was observed which began at the end of viremia. No differences in the response of mice to cloned or uncloned FMDV were observed.

Indirect immunofluorescence and immunodiffusion tests in the detection of antibodies to foot-and-mouth disease virus.

The antibody response detected by indirect immunofluorescence (IIF) as well as that directed against 140 S and virus infection associated antigen (VIA), as detected by agar immunodiffusion, was studied in three mammal species susceptible to Foot and Mouth Disease Virus, after challenge with living virus, immunization and hyperimmunization with inactivated virus, and immunization followed by challenge. By spot indirect immunofluorescence, antibodies were detected only in animals undergoing an active infection, and were not detected in immunized or hyperimmunized animals. This behaviour was similar to that of the anti-VIA antibodies in the same groups of animals and differed from that of anti-140 S antibodies. It appeared that spot indirect immunofluorescence for the detection of VIA antigen is comparable to the immunodiffusion test, but the speed of IIF and the possibility of handling many samples make it more practical.

[Infectious bovine rhinotracheitis (VHB-1). II. Physico-chemical properties of strain L-114]. / Rinotraqueítis infecciosa bovina (VHB-1). II: Propiedades físico-químicas de la cepa L-114.

The thermal stability, density gradient and morphological features of a recently isolated L-114 strain of infectious bovine rhinotracheitis virus were determined. Morphology studies showed an enveloped virus with 200 nm of total diameter, a core diameter of 90 nm and an icosaedral-type structure; purified preparations contained both complete and empty viral particles. More than 90% of the infectivity was lost after 15 hours at 37 degrees C; at 56 degrees C, inactivation was much faster, with a 3 log titer reduction, in 24 minutes. Density gradient studies in cesium chloride, carried out with virus concentrated on sucrose gradient, gave an estimated density of 1.25 g/ml for the purified virus, which fits with light herpesviruses.

Rinotraqueitis infecciosa bovina (VHB-1). II: Propiedades fisico-quimicas de la cepa L-114. / Infectious bovine rhinotracheitis (VHB-1). II.Physico-chemical properties of the L-114 strain

Se determino la estabilidad termica, densidad de flotacion y morfologia de la cepa L-114 de virus de la rinotraqueitis infecciosa bovina, aislada recientemente en el pais. La morfologia correspondio a un virus con envoltura, de 200 mm de diametro total y 90 mm de diametro del "core". La estructura de este ultimo fue de tipo icosaedrica, observandose particulas vacias y completas en la preparaciones purificadas de virus. A 37o C, cepa L-114 perdio mas del 90% de su infectividad en 15 hs. y a 56o C se inactivo muy rapidamente, perdiendo 3 log en 24 minutos. La densidad de flotacion en cloruro de cesio, luego de su concentracion por sedimentacion a traves de sacarosa al 47% (p/v), indica que el virus purificado tiene una densidad estimada de 1,25 g/ml, correspondiente a los virus herpes denominados livianos