- Volume 88, Issue 3, 2007
Volume 88, Issue 3, 2007
- Animal
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- RNA viruses
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Intracellular localization and effects of individually expressed human parechovirus 1 non-structural proteins
More LessHuman parechovirus 1 (HPEV-1) has many unique features compared with other picornaviruses and it has been shown that the replication complex formed during HPEV-1 infection is different from that of other picornaviruses. Here, the intracellular localization and functional effects of individually expressed HPEV-1 non-structural proteins were studied. The 2A and 3D proteins were found diffusely in the cytoplasm and nucleus of the cell. The 3A and 3AB proteins were observed to co-localize with the markers for the Golgi apparatus, whereas 2B co-localized with markers for the endoplasmic reticulum and the 2C and 2BC proteins were observed mainly on the surface of lipid droplets. The 2C protein, which has been implicated in replication-complex formation in enterovirus-infected cells, was not able to induce vesicles similar to those seen in HPEV-1-infected cells when expressed individually. However, in superinfected cells, the fusion protein was able to relocate to the virus replication complexes. Similar to other picornaviruses, HPEV-1 was found to interfere with cellular secretion, but this function could not be ascribed to any of the individually expressed non-structural proteins.
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Protective immune responses in guinea pigs and swine induced by a suicidal DNA vaccine of the capsid gene of swine vesicular disease virus
A suicidal DNA vaccine based on a Semliki Forest virus (SFV) replicon was evaluated for the development of a vaccine against swine vesicular disease virus (SVDV). The 1BCD gene of SVDV was cloned and inserted into pSCA1, an SFV DNA-based replicon vector. The resultant plasmid, pSCA/1BCD, was transfected into BHK-21 cells and the antigenicity of the expressed protein was confirmed using an indirect immunofluorescence assay. Immunogenicity was studied in guinea pigs and swine. Animals were injected intramuscularly three times with pSCA/1BCD at regular intervals. Anti-SVDV antibodies were detected by ELISA, the lymphocyte proliferation response was tested by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide method and neutralizing antibodies were measured by microneutralization tests. The data showed that SVDV-specific antibodies, neutralizing antibodies and lymphocyte proliferation were induced in both guinea pigs and swine. Furthermore, after three successive vaccinations with pSCA/1BCD, half of the pigs were protected against challenge with SVDV. These results should encourage further work towards the development of a DNA vaccine against SVDV.
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Enterovirus 94, a proposed new serotype in human enterovirus species D
The genus Enterovirus (family Picornaviridae) contains five species with strains isolated from humans: Human enterovirus A (HEV-A), HEV-B, HEV-C, HEV-D and Poliovirus. In this study, a proposed new serotype of HEV-D was characterized. Four virus strains were isolated from sewage in Egypt and one strain from acute flaccid paralysis cases in the Democratic Republic of the Congo. The complete genome of one environmental isolate, the complete coding sequence of one clinical isolate and complete VP1 regions from the other isolates were sequenced. These isolates had 66.6–69.4 % nucleotide similarity and 74.7–76.6 % amino acid sequence similarity in the VP1 region with the closest enterovirus serotype, enterovirus 70 (EV70), suggesting that the isolates form a new enterovirus type, tentatively designated enterovirus 94 (EV94). Phylogenetic analyses including sequences of the 5′ UTR, VP1 and 3D regions demonstrated that EV94 isolates formed a monophyletic group within the species HEV-D. No evidence of recombination was found between EV94 and the other HEV-D serotypes, EV68 and EV70. Further biological characterization showed that EV94 was acid stable and had a wide cell tropism in vitro. Attempts to prevent replication with protective antibodies to known enterovirus receptors (poliovirus receptor, vitronectin α v β 3 receptor and decay accelerating factor) were not successful. Seroprevalence studies in the Finnish population revealed a high prevalence of this virus over the past two decades.
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Thermostable variants are not generally represented in foot-and-mouth disease virus quasispecies
More LessA severe limitation to fully realize the dramatic potential for adaptation of RNA virus quasispecies may occur if mutations in vast regions of the sequence space of virus genomes lead to significant decreases in biological fitness. In this study the detection and selection by heat of thermostable variants from different foot-and-mouth disease virus (FMDV) populations were attempted, in order to explore whether FMDV may generally accept a substantial increase in thermostability without compromising its infectivity. The results obtained with both uncloned and cloned populations of different serotypes, recovered from cytolytic or persistent infections and subjected to either very few passages or extensive passaging in cells, indicate that the presence of thermostable virus variants, even in small proportions, is not a general feature of FMDV quasispecies. This suggests that no substantial increase in the thermostability of FMDV may readily occur without a negative effect on viral function.
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Role of the mutant spectrum in adaptation and replication of West Nile virus
West Nile virus (WNV) has successfully spread throughout the USA, Canada, Mexico, the Caribbean and parts of Central and South America since its 1999 introduction into North America. Despite infecting a broad range of both mosquito and avian species, the virus remains highly genetically conserved. This lack of evolutionary change over space and time is common with many arboviruses and is frequently attributed to the adaptive constraints resulting from the virus cycling between vertebrate hosts and invertebrate vectors. WNV, like most RNA viruses studied thus far, has been shown in nature to exist as a highly genetically diverse population of genotypes. Few studies have directly evaluated the role of these mutant spectra in viral fitness and adaptation. Using clonal analysis and reverse genetics experiments, this study evaluated genotype diversity and the importance of consensus change in producing the adaptive phenotype of WNV following sequential mosquito cell passage. The results indicated that increases in the replicative ability of WNV in mosquito cells correlate with increases in the size of the mutant spectrum, and that consensus change is not solely responsible for alterations in viral fitness and adaptation of WNV. These data provide evidence of the importance of quasispecies dynamics in the adaptation of a flavivirus to new and changing environments and hosts, with little evidence of significant genetic change.
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West Nile virus isolates from India: evidence for a distinct genetic lineage
More LessThe complete genomic sequence of one human isolate of West Nile virus (WNV) and the partial genomic sequences of 14 other strains from India isolated in the period 1955–1982 from different hosts and geographical areas were determined. Phylogenetic analyses based on complete and partial genomic sequences (921 nt of the C–prM–E region) revealed that WNV could be classified into five distinct groups that differed from each other by 20–25 % at the complete genome level and by 20–26 % using partial sequences. Of the Indian isolates, 13 formed a distinct genetic lineage, lineage 5, whereas two isolates, one from a human patient (1967) and another from a bat (1968), were related closely to lineage 1 strains. The complete genomic sequence of the Indian isolate, 804994, showed 20–22 % genetic divergence from the previously proposed lineage 1 and 2 strains and 24–25 % divergence from isolates of the newly proposed lineages 3 (Rabensburg isolate 97-103 of 1997) and 4 (Russian isolate LEIV-Krnd88-190 of 1998). Similarly, the partial genomic sequences of the Indian isolates showed 21–26 % divergence from lineage 1 and 2 strains and from the Rabensburg (97-103) and Russian (LEIV-Krnd88-190) isolates. Cross-neutralization using strain-specific polyclonal antibodies against lineage 1 strain Eg-101 and representative Indian strains suggests substantial antigenic variation. This study documents circulation of WNV strains typical to India for 27 years and the introduction of lineage 1 strains during 1967–1968. These results indicate strongly that WNV should be classified into five genetic lineages, with Indian viruses constituting the distinct genetic lineage 5.
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Molecular epidemiological analysis of Japanese encephalitis virus in China
Sixty-two new Japanese encephalitis virus (JEV) isolates were obtained from mosquitoes, biting midges, human cerebrospinal fluid and human blood samples in China during 2002–2005. The E and prM genes were sequenced and phylogenetic analyses were performed with 38 JEV other isolates from China and 36 JEV strains from other countries. Phylogenetic trees based on the E and prM gene sequences were similar. The results indicate that: (i) recent JEV isolates from China are divided into two genotypes, genotype 1 and genotype 3; (ii) recent JEV isolates from China are grouped into the same clusters within genotypes 1 and 3; and (iii) genotype 1 JEV strains have been isolated in China since 1979, whilst genotype 3 JEV strains were isolated before the 1970s. The results suggest that genotype 1 JEV was introduced to China around 1979 and that JEV strains belonging to genotypes 1 and 3 circulate in China.
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A chimeric GB virus B encoding the hepatitis C virus hypervariable region 1 is infectious in vivo
More LessTwo GB virus B (GBV-B) chimeric genomes, GBV-HVR and GBV-HVRh (with a hinge), containing the coding region of the immunodominant hypervariable region 1 (HVR1) of the E2 envelope protein of Hepatitis C virus (HCV) were constructed. Immunoblot analysis confirmed that HVR1 was anchored to the GBV-B E2 protein. To investigate the replication competence and in vivo stability of in vitro-generated chimeric RNA transcripts, two naïve marmosets were inoculated intrahepatically with the transcripts. The GBV-HVR chimeric genome was detectable for 2 weeks post-inoculation (p.i.), whereas GBV-HVRh reverted to wild type 1 week p.i. Sequencing analysis of the HVR1 and flanking regions from GBV-HVR RNA isolated from marmoset serum demonstrated that the HVR1 insert remained unaltered in the GBV-HVR chimera for 2 weeks. Inoculation of a naïve marmoset with serum collected at 1 week p.i. also resulted in viraemia and confirmed that the serum contained infectious particles. All animals cleared the infection by 3 weeks p.i. and remained negative for the remaining weeks. The chimera may prove useful for the in vivo examination of any HCV HVR1-based vaccine candidates.
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Development and evaluation of an efficient cell-culture system for Hepatitis E virus
More LessUsing a faecal suspension with high load of Hepatitis E virus (HEV) (2.0×107 copies ml−1, genotype 3), we developed an efficient cell-culture system for HEV in a hepatocarcinoma cell line (PLC/PRF/5). HEV progeny released in the culture medium were passaged five times successively in PLC/PRF/5 cells. The initial day of appearance and load of HEV detectable in the culture supernatant after inoculation were dependent on the titre of seed virus in the inoculum. When 6.4×104 copies of HEV were inoculated on monolayers of PLC/PRF/5 cells in six-well microplates, HEV RNA was first detected in the culture medium on day 14 post-inoculation and increased to 9.1×105 copies ml−1 on day 60. When 8.6×105 copies of HEV were inoculated, HEV RNA was initially detected on day 12 and reached the highest titre of 8.6×107 copies ml−1 on day 60. HEV incubated at temperatures higher than 70 °C did not grow in PLC/PRF/5 cells, while HEV incubated at 56 °C for 30 min was infectious. Convalescent serum samples with IgM-class HEV antibodies obtained from patients infected with HEV of genotype 1, 3 or 4 neutralized the genotype 3 virus, indicating that HEV antibodies are broadly cross-reactive. Serum samples obtained from patients 8.7 or 24.0 years after the onset of HEV infection also prevented the propagation of HEV in PLC/PRF/5 cells, suggesting the presence of long-lasting HEV antibodies with neutralizing activity in individuals with past HEV infection.
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Detection and characterization of infectious Hepatitis E virus from commercial pig livers sold in local grocery stores in the USA
More LessHepatitis E virus (HEV) is a zoonotic pathogen of which pigs are reservoirs. To determine the presence of HEV RNA in commercial pig livers sold in local grocery stores in the USA, 127 packages of commercial pig liver were purchased and tested by a universal RT-PCR assay capable of detecting all four known HEV genotypes. Among the 127 livers tested, 14 were positive for HEV RNA. Sequence and phylogenetic analyses revealed that the 14 isolates all belonged to genotype 3. An animal study was subsequently conducted in pigs to determine whether the PCR-positive pig livers still contained infectious virus. The results showed that pigs inoculated with two of the three PCR-positive pig-liver homogenates became infected, as evidenced by the detection of faecal virus shedding, viraemia and seroconversion. The data demonstrated that commercial pig livers sold in grocery stores are contaminated by HEV and that the contaminating virus remains infectious, thus raising a public-health concern for food-borne HEV infection.
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Development and characterization of an infectious cDNA clone of the virulent Bucyrus strain of Equine arteritis virus
Strains of Equine arteritis virus (EAV) differ in the severity of the disease that they induce in horses. Infectious cDNA clones are potentially useful for identification of genetic determinants of EAV virulence; to date, two clones have been derived from a cell culture-adapted variant of the original (Bucyrus) isolate of EAV, and it has previously been shown that recombinant virus derived from one of these (rEAV030) is attenuated in horses. A complete cDNA copy of the genome of the virulent Bucyrus strain of EAV has now been assembled into a plasmid vector. In contrast to rEAV030, recombinant progeny virus derived from this clone caused severe disease in horses, characterized by pyrexia, oedema, leukopenia, high-titre viraemia and substantial nasal shedding of virus. The availability of infectious cDNA clones that produce recombinant viruses of different virulence to horses will facilitate characterization of the virulence determinants of EAV through reverse genetics.
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Recombinant swine beta interferon protects swine alveolar macrophages and MARC-145 cells from infection with Porcine reproductive and respiratory syndrome virus
More LessSwine beta interferon (swIFN-β) produced in HEK 293 cells infected with a recombinant, replication-defective human adenovirus 5 (Ad5) encoding the swIFN-β gene was tested for antiviral activity against Porcine reproductive and respiratory syndrome virus (PRRSV). MARC-145 cells were incubated overnight with dilutions of supernatant fluids from HEK 293 cells infected with Ad5-swIFN-β or with an Ad5 control virus (Ad5-Blue). Treated cells were infected with PRRSV; MARC-145 cells incubated with Ad5-Blue supernatants developed cytopathic effects (CPE), whereas those incubated with swIFN-β showed no CPE. To confirm the antiviral activity of swIFN-β, culture fluids from Ad5-swIFN-β-infected cells were affinity-purified on a Sepharose–anti-swIFN-β matrix, and the resulting fractions exhibited antiviral activity upon infection with PRRSV. The antiviral effects were specific, as they were blocked by mAbs against swIFN-β. Additional cultures of MARC-145 cells treated with swIFN-β-containing supernatants or affinity-purified swIFN-β were infected with PRRSV and tested by real-time RT-PCR for viral RNA in culture supernatants at various times post-inoculation. These experiments confirmed the protective effects of swIFN-β. swIFN-β was also tested for antiviral activity on porcine alveolar macrophages (PAMs) obtained by bronchoalveolar lavage from PRRSV-negative swine. PAMs were treated with dilutions of swIFN-β or Ad5-Blue culture fluids, infected with PRRSV and tested for viral RNA by real-time RT-PCR. The viral load data showed a dose-dependent protection in swIFN-β-treated PAMs, whereas no protection was evident from Ad5-Blue culture fluids. The data demonstrate that swIFN-β protects both MARC-145 cells and PAMs from PRRSV infection.
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Genomic analysis of diverse rubella virus genotypes
More LessBased on the sequence of the E1 glycoprotein gene, two clades and ten genotypes of Rubella virus have been distinguished; however, genomic sequences have been determined for viruses in only two of these genotypes. In this report, genomic sequences for viruses in an additional six genotypes were determined. The genome was found to be well conserved. The viruses in all eight of these genotypes had the same number of nucleotides in each of the two open reading frames (ORFs) and the untranslated regions (UTRs) at the 5′ and 3′ ends of the genome. Only the UTR between the ORFs (the junction region) exhibited differences in length. Of the nucleotides in the genome, 78 % were invariant. The greatest observed distance between viruses in different genotypes was 8.74 % and the maximum calculated genetic distance was 14.78 substitutions in 100 sites. This degree of variability was similar among regions of the genome with two exceptions, both within the P150 non-structural protein gene: the N-terminal region that encodes the methyl/guanylyltransferase domain was less variable, whereas the hypervariable domain in the middle of the gene was more divergent. Comparative phylogenetic analysis of different regions of the genome was done, using sequences from 43 viruses of the non-structural protease (near the 5′ end of the genome), the junction region (the middle) and the E1 gene (the 3′ end). Phylogenetic segregation of sequences from these three genomic regions was similar with the exception of genotype 1B viruses, among which a recombinational event near the junction region was identified.
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Effect of the phosphatidylinositol 3-kinase/Akt pathway on influenza A virus propagation
More LessThe phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway has attracted much recent interest due to its central role in modulating diverse downstream signalling pathways associated with cell survival, proliferation, differentiation, morphology and apoptosis. An increasing amount of information has demonstrated that many viruses activate the PI3K/Akt pathway to augment their efficient replication. In this study, the effect of the PI3K/Akt signalling pathway on influenza virus propagation was investigated. It was found that Akt phosphorylation was elevated in the late phase of influenza A/PR/8/34 infection in human lung carcinoma cells (A549). The PI3K-specific inhibitor LY294002 could suppress Akt phosphorylation, suggesting that influenza A virus-induced Akt phosphorylation is PI3K-dependent. UV-irradiated influenza virus failed to induce Akt phosphorylation, indicating that viral attachment and entry were not sufficient to trigger PI3K/Akt pathway activation. Blockage of PI3K/Akt activation by LY294002 and overexpression of the general receptor for phosphoinositides-1 PH domain (Grp1-PH) led to a reduction in virus yield. Moreover, in the presence of LY294002, viral RNA synthesis and viral protein expression were suppressed and, possibly as a consequence of low NP and M1 protein level, viral RNP nuclear export was also suppressed. These data suggest that the PI3K/Akt signalling pathway plays a role in influenza virus propagation.
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Antibodies specific to the HA2 glycopolypeptide of influenza A virus haemagglutinin with fusion-inhibition activity contribute to the protection of mice against lethal infection
More LessFour monoclonal antibodies (mAbs) recognizing distinct antigenic sites on the HA2 glycopolypeptide of influenza virus A/Dunedin/4/73 (H3N2) have been tested for in vivo protection. When applied intravenously before infection, three of them increased the survival of BALB/c mice infected with 1 LD50 homologous virus. The protection resulted simultaneously in 2 days earlier clearance of virus from the lungs. These three antibodies inhibited the fusion activity of virus in previous in vitro experiments. One of them, specific to N-terminal aa 1–38 of the HA2 glycopolypeptide, was also tested for protection against the heterologous virus A/Mississippi/1/85 (H3N2). Protection similar to that against the homologous virus was observed. The fourth mAb, without fusion-inhibition activity, did not protect mice. It is concluded that antibodies specific to the antigenically conserved HA2 glycopolypeptide that exhibit fusion-inhibition activity can contribute to the protection of infected mice and mediate more effective recovery from infection.
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Mapuera virus, a rubulavirus that inhibits interferon signalling in a wide variety of mammalian cells without degrading STATs
More LessMapuera virus (MPRV) is a paramyxovirus that was originally isolated from bats, but its host range remains unknown. It was classified as a member of the genus Rubulavirus on the basis of structural and genetic features. Like other rubulaviruses it encodes a V protein (MPRV/V) that functions as an interferon (IFN) antagonist. Here we show that MPRV/V differs from the IFN antagonists of other rubulaviruses in that it does not induce the proteasomal degradation of STAT proteins, key factors in the IFN signalling cascade. Rather, MPRV/V prevents the nuclear translocation of STATs in response to IFN stimulation and inhibits the formation of the transcription factor complex ISGF3. We also show that MPRV/V blocks IFN signalling in cells from diverse mammalian species and discuss the IFN response as a barrier to cross-species infections.
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Identification of novel canine rabies virus clades in the Middle East and North Africa
Four novel phylogenetic clades of canine rabies virus (RABV) variants have been identified in the Middle East and North Africa. The three novel Middle Eastern clades comprise RABV isolates from the borders between Israel and neighbouring countries. The North African clade (Africa 4) comprises four RABV isolates from Egypt and one from Israel. We characterized various RABV lineages antigenically by using a panel of monoclonal antibodies to the nucleoprotein (N) and phylogenetically by analysis of entire N gene sequences. The estimated mean substitution rate for the N gene alignment (2.7×10−4 substitutions per site per year) is comparable with previous estimates for RABV. The application of a molecular clock indicates the emergence of current canine RABV diversity to have occurred at about the same time (approx. 1870) in the Middle East and Europe, following divergence from established lineages in Africa and Asia.
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Thioaptamer decoy targeting of AP-1 proteins influences cytokine expression and the outcome of arenavirus infections
Viral haemorrhagic fever (VHF) is caused by a number of viruses, including arenaviruses. The pathogenesis is believed to involve dysregulation of cytokine production. The arenaviruses Lassa virus and Pichinde virus have a tropism for macrophages and other reticuloendothelial cells and both appear to suppress the normal macrophage response to virus infection. A decoy thioaptamer, XBY-S2, was developed and was found to bind to AP-1 transcription factor proteins. The P388D1 macrophage-like cell line contains members of the AP-1 family which may act as negative regulators of AP-1-controlled transcription. XBY-S2 was found to bind to Fra-2 and JunB, and enhance the induction of cytokines IL-6, IL-8 and TNF-α, while reducing the binding to AP-1 promoter elements. Administration of XBY-S2 to Pichinde virus-infected guinea pigs resulted in a significant reduction in Pichinde virus-induced mortality and enhanced the expression of cytokines from primary guinea pig macrophages, which may contribute to its ability to increase survival of Pichinde virus-infected guinea pigs. These data demonstrate a proof of concept that thioaptamers can be used to modulate the outcome of in vivo viral infections by arenaviruses by the manipulation of transcription factors involved in the regulation of the immune response.
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Phylogenetic analysis of Heliothis armigera cytoplasmic polyhedrosis virus type 14 and a series of dwarf segments found in the genome
More LessFull-length nucleotide sequences for the genome segments (S1–S6) of Heliothis armigera cytoplasmic polyhedrosis virus type 14 (HaCPV-14) have been characterized. Each segment consists of a single open reading frame with conserved motifs AGAA and AGCU at the 5′ and 3′ ends, respectively. Comparison of the proteins of HaCPV-14 with those of other members of the family Reoviridae suggests that S1 encodes an RNA-dependent RNA polymerase (RdRp), whilst S2 encodes a major capsid protein of the virus. Phylogenetic analysis of RdRps from 16 viruses in the family Reoviridae reveals that the genera Cypovirus and Oryzavirus may have originated from a common insect virus ancestor. A series of viable dwarf segments originating from S5 of HaCPV-14 has been identified. Analysis of the predicted secondary structures for these dwarf segments suggests that the signals essential for replication and packaging are located within the terminal sequences of these segments.
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Correlation between the induction of Th1 cytokines by an attenuated equine infectious anemia virus vaccine and protection against disease progression
More LessThe equine infectious anemia virus (EIAV) donkey-leukocyte attenuated vaccine (DLV) has been used to protect against equine infectious anaemia (EIA) disease for several decades in China. The attenuated mechanism and immunological protective mechanisms remain to be elucidated. To identify responses that correlate with the protection against disease, we immunized horses with DLV, followed by challenge with an EIAV wild-type strain LN. All vaccinated horses were asymptomatic and had a low level of virus replication (<10 copies ml−1). The expression level of cytokines including gamma interferon, interleukin 2 and 12 in DLV immunized horses was 5–100-fold higher than that in non-vaccinated controls (n=4, P<0.01). After challenge with virulent LN, horses vaccinated with DLV showed lower viral loads (<103 copies ml−1) with no temperature increase, except for one transient febrile episode in one animal. In contrast, horses in the non-vaccinated control group experienced much higher viral loads (>107 copies ml−1) and intermittent febrile episodes. Cytokine production in the DLV-vaccinated horses increased and attained a plateau level at approximately 50 days post-vaccination, and exceeded 107 copies per 107 peripheral blood mononuclear cells (PBMCs) 1–3 months post-challenge. However, non-vaccinated control horses died after several fever episodes (⩾39 °C), which coincided with higher viral load (106–107 copies ml−1) and lower cytokine production (<104 copies per 107 PBMCs). The results indicate that high levels of EIAV-specific cytokines induced by the attenuated EIAV vaccine may contribute to the protective immune response against EIA disease.
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Volumes and issues
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Volume 105 (2024)
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