This study showed that asymptomatic, replicative Ebola infection can and does occur in human beings. The lack of genetic differences between symptomatic and asymptomatic individuals suggest that asymptomatic Ebola infection did not result from viral mutations. Elucidation of the factors related to the genesis of the strong inflammatory response occurring early during the infectious process in these asymptomatic individuals could increase our understanding of the disease.

11 of 24 asymptomatic individuals developed both IgM and IgG responses to Ebola antigens, indicating viral infection. Western-blot analysis showed that IgG responses were directed to nucleoprotein and viral protein of 40 kDa. The glycoprotein and viral protein of 24 kDa genes showed no nucleotide differences between symptomatic and asymptomatic individuals. Asymptomatic individuals had a strong inflammatory response characterised by high circulating concentrations of cytokines and chemokines.

Blood was collected from 24 close contacts of symptomatic patients. These asymptomatic individuals were sampled 2, 3, or 4 times during a 1-month period after the first exposure to symptomatic patients. Serum samples were analysed for the presence of Ebola antigens, virus-specific IgM and IgG (by ELISA and western blot), and different cytokines and chemokines. RNA was extracted from peripheral blood mononuclear cells, and reverse-transcriptase-PCR assays were done to amplify RNA of Ebola virus. PCR products were then sequenced.

Ebola virus is one of the most virulent pathogens, killing a very high proportion of patients within 5–7 days. Two outbreaks of fulminating haemorrhagic fever occurred in northern Gabon in 1996, with a 70% case-fatality rate. During both outbreaks we identified some individuals in direct contact with sick patients who never developed symptoms. We aimed to determine whether these individuals were indeed infected with Ebola virus, and how they maintained asymptomatic status.

Two outbreaks of Ebola virus occurred in northern Gabon in early and late 1996, resulting in case-fatality rates of 66% and 75% among 59 and 60 symptomatic patients with laboratory-confirmed infections.Most patients developed high fever, headache, diarrhoea, vomiting, and haemorrhagic manifestations. The virus was transmitted from a dead chimpanzee in the first outbreak and from person to person in the second outbreak, via infected body fluids(faeces, vomit, saliva, sweat, or blood). From the beginning to the end of both outbreaks, we identified 24 individuals who were directly exposed to infected materials from fatal and non-fatal cases, but who did not develop symptoms. These individuals were family members of symptomatic patients who lived continuously with them, and took care of them without any physical protection such as gloves. Throughout the study, these individuals were sampled several times, from the time of first exposure to a sick patient. We report a description of asymptomatic and replicative Ebola infection in human beings.

We have found that immunological events very early in an Ebola-virus infection determine the control of viral replication and recovery or catastrophic illness and death.Recovery from infection is related to orderly and well-regulated humoral and cellular immune responses, characterised by the early appearance of IgM and IgG, followed by activation of cytotoxic cells at the time of antigen clearance from blood. By contrast, fatal outcome is associated with impaired humoral responses and an early activation of T cells unable to control virus replication, followed by considerable intravascular apoptosis.

Ebola virus belongs to the Filoviridae family and is subdivided into four subtypes: Zaire, Sudan, Côte d'Ivoire, and Reston.Ebola virus is one of the most virulent pathogens, killing a very high proportion of patients within 5–7 days.The virus is endemic in central Africa, where it occasionally causes fulminating haemorrhagic disease in human and non-human primates.The genome is composed of linearly arranged genes on a single negative-stranded RNA molecule that encodes the seven structural proteins: nucleoprotein, virion structural proteins VP35, VP40, glycoprotein (GP), VP30 and VP24, and RNA-dependent RNA polymerase (L).The organisation and transcription of the GP gene is unusual and involves transcriptional editing before it can be expressed.

The virion glycoproteins of Ebola viruses are encoded in two reading frames and are expressed through transcriptional editing.

Plasma concentrations of interleukin-1β (IL-1β), interferon alpha (IFNα), I, IL-12, IL-13, tumour necrosis factor (TNF), macrophage inflammatory protein-1α (MIP-1α), and MIP-1β were measured with commercial kits (R&D Systems Europe, Abingdon, UK). Plasma concentrations of IL-10 and interferon gamma (INFγ) were each measured at the same time with two different kits (IL-10: Amersham and Immunotech, Orsay and Marseille, France; IFNγ: R&D Systems Europe and Immunotech). Plasma concentrations of the other cytokines were measured with a two-site ELISA, with specific capture and biotinylated antibodies against IL-2 or IL-6 (Genzyme, Cergy-St-Christophe, France), IL-4, IL-5 (Pharmingen, CA, USA). To confirm the results, plasma concentrations of IL-2, IL-4, and IL-6 were also measured with commercial kits (Immunotech). Optical density was measured at 492 mm on an ELISA plate reader (Diagnostics Pasteur). Cytokine concentrations were calculated from standard curves. Samples from all patients were taken, treated, and stored in exactly the same conditions. In patients who displayed symptoms, the mean time from infection was assessed on the basis of the time of the earliest samples after the onset of symptoms, and the mean incubation period. Because the incubation period is between 5–8 days and that the earliest samples were taken within 3 days after the onset of symptoms, the samples from symptomatic patients were from 8–11 days after the putative infection.

The PCR fragment of 298 bp from the L gene was purified and sequenced with the Sequenase II kit (Amersham). Reverse transcription and first and second round PCR amplification of VP24 and GP were done for the L gene. For VP24 we used 5′-ACATCACTTTGAGCGCCCTCA-3′ and 5′-AGCTGG CTTACAGTGAGGATT-3′ as primers for the first round and 5′-GCGCAAGGTTTCAAGGTTGAA-3′ and 5′-TTGAGT CAGCATATATGAGTT-3′ as primers for the second round of PCR. For amplification of GP, we needed to divide it into two overlapping parts of equal size which represent together the complete open reading frame. Primers for the first part were: 5′-GATGAAGATTAAGCCGACAGTGAGCG-3′ and 5′-GTTGTCTGTTCTGCGGTGATGTTG-3′ for the first round of PCR and 5′-GTGAGCGTAATCTTCA TCTCTCTTAG-3′ and 5′-TTGTTCAACTTGAGTTGCCTCAGAG-3′ for the second round. Primers for the second part were: 5′-TGCAATGGTTCAAGTGCACAGTC-3′ and 5′-AAGAGATAACTAGAT TGATGTCAAACC-3′ for the first round and 5′-CAAGGAAGGGAAGCTGCAGTGTCG-3′ and 5′-GAATCACATTGGCTATGTTTAAAGC-3′ as primers for the second round. The different amplification products were electrophoresed on 1·5% agarose gels, stained with ethidium bromide, excised from the gel, and extracted with a QIA quickgel extraction kit (Qiagen, Courtaboeuf, France). Sequencing of VP24 fragments was run on an ALF express DNA sequencer (Pharmacia Biotech, Uppsala, Sweden) with an Autocycle 200 sequencing kit (Pharmacia). Analysis was done with the OS/2 computer system. Sequencing of GP fragments was done by ACTgene laboratory (ACTgene, Evry, France).

Peripheral blood mononuclear cells were separated from whole blood by Ficoll-diatrizoate density-gradient centrifugation. Total RNA was extracted with a kit (Qiagen, Courtaboeuf, France). The first-strand complementary (cDNA) was synthesised by the superscript II kit (Gibco BRL, Eragny, France), a dNTP mix (Amersham, Orsay, France), random hexamer primers (PCR1, PCR2; Boehringer, Mannheim, Germany), primers specific for positive-strand RNA, or primers specific for negative-strand RNA. Half of the reaction product was used as a template for PCR with Taq DNA polymerase (Appligene-Oncor, Illkirch, France) for 40 cycles (94°C for 30 s, 55°C for 30 s, and 72°C for 90 s). The reaction was run in a Perkin-Elmer 480 thermocycler (Perkin Elmer, Rotkreuz, Switzerland) with designed primers from a conserved region of the L gene (coding for RNA-dependent RNA polymerase), as described elsewhere.The amplification products were analysed on 1·5% agarose gels. After the first round of amplification, the products of interest were identified by southern hybridisation. The 420 bp reverse transcriptase PCR products were purified and labelled with a digoxigenin labelling kit (Boehringer). The PCR products were then run in 1·5% agarose gel and transferred overnight to a positively charged nylon membrane with 1 M sodium hydroxide. Hybridisation and chemiluminescence detection were done using the Dig kit (Boehringer). For nested PCR, 5 μL of the first-round reaction product was used as a template, and the reaction was run in the same conditions as first-round PCR. The 298 bp products from the nested PCR (PCR2) were also run in 1·5% agarose gel.

Purified Ebola virus from tissue culture medium was loaded on 10% sodium dodecyl sulfate-polyacrylamide gel and transferred onto a polyvinylidene fluoride membrane at 55 V for 3 h at 4°C. Protein immunoblotting analysis was done with serum samples diluted in a ratio of 1:500, and a secondary antibody (goat antibody to human IgG peroxidase conjugate) in a dilution of 1:30 000. All incubations were done for 1 h at room temperature. Immune complexes were visualised by chemiluminescence detection with Super Signal Substrate reagents (Pierce, Rockford, IL, USA) according to the manufacturer's instructions.

Results were interpreted as previously described.A panel of ten endemic normal serum samples was run each time the assay was used. Adjusted optical densities were calculated by subtracting the optical density of uninfected antigen-coated well from its corresponding antigen-coated well. The cut-off value was given as the mean adjusted optical density for the ten normal serum samples (+350). All samples were handled according to WHO guidelines on viral haemorrhagic fever in Africa (WHO recommendations for management of viral haemorrhagic fevers in Africa, Sierra Leone, 1985).

The IgG assay we used was an ELISA in which plates were coated with Ebola Z antigen, diluted 1:1000 in phosphate buffered saline (PBS), and incubated overnight at 4°C. Plates were coated with uninfected vero cell culture antigens under the same conditions. Serum samples were then diluted 1:400, 1:1600, 1:3200, 1:6400 in 5% non-fat milk in PBS-Tween 20 and incubated overnight at 4°C. Binding was visualised with peroxidase-labelled antibody to human IgG (Sigma, L'Isle d'Abeau, France) and the TMB detector system (Dynex Technologies, Issy-les-Moulineaux, France). Optical density was measured at 450 nm, on an ELISA plate reader (Diagnostics Pasteur, Marne la Coquette, France).

We used the standard IgM capture assay (carried out by Special Pathogens Branch, CDC, Atlanda, GA, USA).IgM from the serum sample of the infected patients were first captured with antibody to human IgM. Viral antigens were then added to the captured IgM and were exposed to a polyclonal hyperimmune rabbit serum containing antibodies to Ebola virus. Bound antibodies to Ebola virus were detected by antibody to rabbit IgG conjugated to peroxidase.

By contrast, no IFNα, no IL-12, and no T-cell-derived cytokines (IL-2, IL-4, IL-5, or IFNγ) were detected in the plasma of asymptomatic individuals at any time during the sampling period.

High concentrations of pro-inflammatory cytokines IL-6, IL-1β, and TNF and chemokines MCP-1, MIP-1α, and MIP-1β were detected 1 week after the first potentially infectious contact, whereas concentrations were below the detection limit in uninfected controls, indicating a strong inflammatory response triggered 4–6 days after infection ( figure 3 ). The inflammatory responses in the asymptomatic individuals disappeared rapidly within just 2–3 days, thereby avoiding fever and other physiological disturbances indicating tissue or organ damage.

Concentrations of pro-inflammatory cytokines (IL-1β, TNF, and IL-6, expressed in ng/L) and chemokines (MCP-1, MIP-1α, and MIP-1β, in μg/L) were measured in plasma by ELISA. Individual values for the seven PCR-positive asymptomatic individuals are shown between 7 and 23 days after the first potentially infectious contact. Each asymptomatic individual is represented by a point.

Amplicons of 853 bp (VP24) and 2445 bp (GP) were obtained in three fatal cases, three non-fatal symptomatic cases, and three asymptomatic individuals. Only second-round PCR yielded the fragments of the expected size in samples from asymptomatic individuals, whereas in symptomatic patients one-step PCR was sufficient, indicating once again that viral RNA is present in low copy numbers in asymptomatic individuals. Comparative sequence analysis of the GP gene showed 36 nucleotide substitutions that led to 15 switches in the predicted aminoacid sequences in Gabon-96 Ebola virus relative to Zaire Mayinga 76. By contrast, analysis of VP24 showed only eight nucleotide substitutions in Gabon-96 relative to Zaire Mayinga 76, but none of these changes led to switches in the deduced VP24 aminoacid sequences. Isolates from all nine patients tested had identical VP24 and GP nucleotide sequences.

To find out how long viral RNA could be detected in asymptomatic individuals, we sampled two of the seven such individuals on four occasions ( figure 2 ). We detected viral genomic RNA for up to 2 weeks after the last known exposure to infected materials, suggesting that the virus replicated in the patients' mononuclear cells. To confirm this, we used reverse transcriptase PCR with primer specific for positive-strand RNA. As described in figure 2 , four of the seven asymptomatic patients were positive, confirming virus replication because Ebola virus (EBOV) is a negative-strand virus. Interestingly, the positive-strand RNA signal disappeared between 9 and 16 days after the first infectious contactie, 1 week before the detection of specific antibodies, indicating transient virus replication that lasted about 2 weeks.

We developed a reverse-transcriptase PCR assay to detect viral RNA fragment in the L gene in peripheral blood mononuclear cells with primers. The PCR assay was negative in all these individuals, whereas the appropriate 420 bp fragment was detected in all symptomatic patients infected with Ebola virus ( figure 2 ). By contrast, second-round PCR yielded DNA products of the expected size (298 bp) in seven of 11 asymptomatic individuals tested but in none of the 13 of the exposed antibody-negative individuals and in none of the negative control individuals ( figure 2 ). Southern-blot hybridisation of the first-round PCR products was also positive ( figure 2 ). The viral specificity of the nested PCR assay was confirmed by sequencing the amplicon (data not shown).

A Results for seven asymptomatic individuals are shown. They were sampled between 7 and 16 days after the first exposure to a sick patient. The 420 bp product obtained from the first round of PCR (PCR 1) and the 298 bp product obtained after two steps of PCR (PCR 2) represent cDNA fragments of the Ebola-virus polymerase gene. The first-strand cDNA was synthesised by random hexamer primers (PCR1, PCR2) or primers specific for positively-stranded RNA (PCR2+) or primers specific for negative-stranded RNA (PCR2-). (+) is a positive symptomatic patient during the acute phase of the disease. (-) is a healthy negative endemic control. (M-) is PCR mix without cDNA. These controls were included in each run. Ten negative controls were tested but only one is shown in the figure. B Detection of Ebola RNA during infection. The cDNA was constructed with a primer set specific for negative-stranded RNA (PCR-) or positive-stranded RNA (PCR+). Peripheral-blood mononuclear cells were collected from two asymptomatic individuals (asymptomatic 1 and 2), 7, 9, 16, and 23 days after the first exposure to a sick patient.

By contrast with the symptomatic patients, circulating Ebola antigen was never detected by an antigen capture assay in serum samples from antibody-positive patients. Similarly, we did not isolate virus on vero-E6 cells, without blind passages, from serum from the seven individuals.

We investigated these apparently protected individuals by measuring viral-specific IgM and IgG in serum samples collected serially at various times during the two outbreaks, with Ebola Zaire antigens ( figure 1 ). The first samples from each individual were without antibody, excluding prior immunity. 11 of the 24 patients tested later developed both IgM and IgG responses to viral antigens. Concentrations of specific IgM started to increase 15–18 days after the first identified potentially infectious contact, and about 10 days after the last such contact ( figure 1 ). 1 month after the last exposure, Ebola-specific IgM was found in all 11 individuals. Virus-specific IgG appeared 1 week after IgM, and reached lower titres than IgM. When serum samples from IgG-positive asymptomatic individuals were analysed by western blotting in denaturing conditions with purified Ebola virus, reactivity was mainly directed against the nucleoprotein (NP) or VP40 proteins ( figure 1 ).

Specific IgM and IgG antibody responses to Ebola Zaire, as determined by ELISA. Each point represents one contact individual who was sampled several times.Antibody responses analysed by western blotting for one symptomatic patient who recovered (T+), two negative endemic controls (EC), and five IgG-positive asymptomatic individuals. Identity of each band from symptomatic case was confirmed previously with antibody to Zaire hyperimmune goat serum.

Discussion

Despite our inability to isolate Ebola virus on vero E6 cells from stored serum from the seven asymptomatic individuals, these data do show that asymptomatic, replicative Ebola-virus infection occurs in human beings.

7 Baize S

Leroy EM

Georges-Courbot M-C

et al. Defective humoral responses and extensive intravascular apoptosis are associated with fatal outcome in Ebola virus-infected patients. 11 Xu L

Sanchez A

Yang Z-Y

et al. Immunization for Ebola virus infection. 7 Baize S

Leroy EM

Georges-Courbot M-C

et al. Defective humoral responses and extensive intravascular apoptosis are associated with fatal outcome in Ebola virus-infected patients. First, these individuals mounted Ebola-specific humoral responses, mainly directed against the proteins NP and VP40, indicating either true infection or antigenic stimulation. Concentrations of IgM and IgG started to rise between 10 and 18 days and between 17 and 25 days, respectively, after the infectious contact, indicating no prior immunity. The appearance of IgM and IgG when viral RNA disappeared may suggest that humoral responses can have a protective role in asymptomatic infection. These findings also confirmed the immunogenicity of NP and VP40 and were consistent with results obtained in symptomatic human beings who survived the diseaseand with other results obtained in animal studies.The appearance of antibodies came somewhat later in asymptomatic patients than in symptomatic patients who recovered(day 3 following disease onset, about 8–11 days after presumed exposure assuming a 5–8 day incubation period).

10 Sanchez A

Ksiazek TG

Rollin PE

et al. Detection and molecular characterization of Ebola viruses causing disease in human and nonhuman primates. 12 Feldmann H

Bugany H

Mahner F

Klenk HD

Drenckhahn D

Schnittler HJ Filovirus-induced endothelial leakage triggered by infected monocytes/macrophages. 13 Geisbert TW

Jahrling PB

Hanes MA

Zack PM Association of Ebola-related Reston virus particles and antigen with tissue lesions of monkeys imported to the United States. , 14 Bray M

Davis K

Geisbert T

Schmaljohn C

Huggins J A mouse model for evaluation of prophylaxis and therapy of Ebola hemorrhagic fever. 15 Leroy EM

Baize S

Lu C-Y

et al. Diagnosis of Ebola haemorrhagic fever by RT-PCR in an epidemic setting. 1 Feldmann H

Klenk HD Marburg and Ebola viruses. Despite seroconversion, circulating Ebola antigen was never detected in asymptomatic individuals. Then, to lower the detection threshold and to distinguish passive immunisation from true infection in the asymptomatic individuals, we developed a reverse-transcriptase PCR assay to detect viral RNA fragment in the L gene in peripheral-blood mononuclear cells with primers described previously.We tested circulating white blood cells rather than serum because filoviruses replicate in cells of the monocyte and macrophage lineage both in vitroand in vivo.Ebola RNA was detected in samples from asymptomatic individuals after two rounds of amplification. The need to apply nested PCR to detect viral RNA in these asymptomatic individuals compared with a direct PCR in symptomatic casesis suggestive of a very low viral load, consistent with the absence of detectable circulating antigens. Moreover, detection of viral genomic RNA in peripheral-blood mononuclear cells for 2 weeks after exposure together with detection of positive-stranded viral RNA indicate viral replication. In fact, it has been shown that the Ebola genome is transcribed into monocistronic RNA (mRNA), which is complementary to viral genomic RNA. Replication works via a full-length positive-strand antigenome that serves as the template for synthesis of the negative-strand genome.

16 Smith DIH Ebola haemorrhagic fever in Sudan, 1976. 17 Johnson KM Ebola haemorrhagic fever in Zaire, 1976. , 18 Baron RC

McCormick JB

Zubeir OA Ebola virus disease in southern Sudan: hospital dissemination and intrafamilial spread. 19 Rowe AK

Bertolli J

Khan AS

et al. Clinical, virologic, and immunologic follow-up of convalescent Ebola hemorrhagic fever patients and their household contacts, Kikwit, Democratic Republic of the Congo. These findings show that some individuals were infected with the virus without developing symptoms. Results from previous outbreaks had only indicated that such an asymptomatic infection was possible. During the first three outbreaks of Ebola virus in Sudan and Zaire in 1976 and 1979, WHO teams noticed that individuals had symptoms that ranged in severity, from mild (and probably asymptomatic) to rapidly fatal.Moreover, the immunofluorescence showed higher antibody prevalence among asymptomatic family members who had had physical contact with clinical cases than among the general population who had no contact with symptomatic patients.More recently, a cohort of 152 household contacts of convalescents was studied for up to 21 months during the Kikwit outbreak in Republic Democratic of the Congo.Blood samples of only five such individuals were IgM and IgG positive. Although the authors could not exclude the possibility of false positive (5 [3%] of 152), they suggested that mild cases may occur.

14 Bray M

Davis K

Geisbert T

Schmaljohn C

Huggins J A mouse model for evaluation of prophylaxis and therapy of Ebola hemorrhagic fever. , 20 Ryabchikova E

Kolesnikova L

Smolina M

et al. Ebola virus infection in guinea pigs: presumable role of granulomatous inflammation in pathogenesis. 4 Feldmann H

Klenk HD

Sanchez A Molecular biology and evolution of filoviruses. 21 Yang Z-Y

Delgado R

Zu L

et al. Distinct cellular interactions of secreted and transmembrane Ebola virus glycoproteins. Interestingly, guinea pigs and mice develop a very mild or subclinical infection when inoculated with wild-type Ebola virus, but serial passage leads to increasing pathogenicity and high lethality.Genomic sequence analysis of the entïre genome of guinea-pig-adapted Ebola virus showed several nucleotide substitutions, mainly in the VP24 gene; only changes in VP24 led to switches in the predicted aminoacid sequence, suggesting a direct link between VP24 mutations and appearance of pathogenicity of the virus (Volchkov, unpublished data). Furthermore, the GP protein is known to be involved in the virus binding to cellular receptors and entry into cells,and is suspected to inhibit innate immunity to the virus, facilitating virus replication and symptoms.Thus, the complete open reading frame of these two genes was sequenced by nested-PCR amplification.

22 Rodriguez LL

De Roo A

Guimard Y

et al. Persistence and genetic stability of Ebola virus during the outbreak of Kikwit, Democratic Republic of the Congo, 1995. The nucleotide divergence between GP and VP24 occurring in Yambuku (Mayinga strain) and Ebola in Gabon was found to be 1·5% and 0·5%, respectively, despite the fact that they were isolated more than 20 years and almost 2000 km apart. This confirms the genetic stability of the virus. Moreover, isolates from all nine patients tested had identical GP and VP24 nucleotide sequences. These findings suggest genetic variability between symptomatic (survivors and deceased patients) and asymptomatic individuals, and are consistent with the lack of changes seen in the most variable region of GP during repeated human-to-human passages and during prolonged virus persistence within the same patients in the Kikwit outbreak in 1995.These data suggest that asymptomatic Ebola infection in human beings does not result from viral mutations, and that no different virus variants cocirculated during the Gabon outbreak.

23 Vassilli P The pathophysiology of tumor necrosis factors. , 24 Dinarello CA Biologic basis for interleukin-1 in disease. 25 Taub DD

Conlon K

Lloyd AR

Oppenheim JJ

Kelvin DJ Preferential migration of activated CD4+ and CD8+ T cells in response to MIP-1α and MIP-1ß. , 26 Gunn MD

Nelken NA

Liao X

Williams LT Monocyte chemoattractant protein-1 is sufficient for the chemotaxis of monocytes and lymphocytes in transgenic mice but requires an additional stimulus for inflammatory activation. 27 Orange JS

Biron CA Characterization of early IL-12, IFN-αß, and TNF effects on antiviral state and NK cell responses during murine cytomegalovirus infection. , 28 Ruby J

Bluethmann H

Peschon JJ Antiviral activity of tumor necrosis factor (TNF) is mediated via p55 and p75 TNF receptors. 29 Le JM

Vilcek J Accessory function of human fibroblasts in mitogen-stimulated interferon-gamma production by T-lymphocytes. Inhibition by interleukin 1 and tumor necrosis factor. , 30 Unanue ER

Allen PM The basis for the immunoregulatory role of macrophages and other accessory cells. The seven asymptomatic individuals in our study had an early and strong inflammatory response with high circulating levels of IL-1β, TNF, IL-6, MCP-1, MIP-1α, MIP-1β ( figure 3 ). IL-1β, IL-6, and TNF have been shown to be major triggers and regulators of inflammatory responses to microbial pathogens, inducing further cytokine release, endothelial cell activation, acute-phase proteins synthesis, and fever.MCP-1, MIP-1α, and MIP-1β have a role in the recruitment of immune cells to the site of infection (being potent chemoattractants for monocytes and lymphocyte effector cells), and also enhance the capacity of these cells to adhere to the vascular endothelium.The pro-inflammatory cytokine response in the asymptomatic individuals might have directly or indirectly inhibited viral repliation in its target cells, probably at the site of infection. Indeed, the cytokines involved have been shown to inhibit virus replication either directlyor indirectly by stimulating immunological functions such as antigen presentation, cytokine production, inflammation, phagocytosis, and cytotoxic activity.