Respiratory viruses associated with patients older than 50 years presenting with ILI in Senegal, 2009 to 2011.

BACKGROUND: In Africa, especially in West Africa, studies about the prevalence and diversity of respiratory viruses (influenza and others) in elderly people are largely lacking. In studies done elsewhere, it is well established that older people, when compared with younger adults, are at greater risk of significant morbidity and mortality from complications arising from influenza. The main aim of this study was to determine the prevalence and the diversity of respiratory viruses associated with ILI cases in adults over 50 years old in Senegal. METHODS: The recruitment period of this study was from January 2009 to December 2011. 232 patients aged 50 years and above presenting ILI cases were enrolled. Nasal-pharyngeal and/or oral pharyngeal swabs were collected from patients. RNA was extracted from 200 µl of each sample followed by a two-step real-time RT-PCR. The Anyplex™ II RV16 Detection kit was used for viral detection. The kit enabled the simultaneous detection of the presence of 16 respiratory viruses. RESULTS: 150 viruses were detected: influenza viruses (44.7%) and rhinoviruses (26.7%) were the most prevalent. We detected 13 human parainfluenza viruses (8.7%), 7 human respiratory syncytial viruses (4.7%), 6 coronaviruses (4%), 5 human metapneumoviruses (3.3%), 5 human adenoviruses (3.3%) and 1 human bocavirus (0.7%). 14 cases (6%) of dual virus infections and one triple viral detection case were encountered. 56 (56.6%) viruses detected were found in the 50-64 year old age group, 59 (76.6%; P < 0.001) from 65-74 year old age group and 35 (62.5%) were detected in the ≥75 year old age group. The viral co-infections were more frequent in the 65-74 age group (9/15). CONCLUSIONS: This pilot study demonstrates a variety of respiratory viruses in the elderly. It also highlights a high prevalence of these viruses in this age group. We speculate from these results that the impact of respiratory viruses other than influenza on the elderly has been considerably underestimated. A more exhaustive study seems necessary in order to provide a more complete picture of the burden of respiratory viruses on morbidity among adults over 50 years old in the sub-Saharan context.

Spread of Influenza A(H1N1) oseltamivir-resistant viruses in Africa in 2008 confirmed by multiple introductions in Senegal.

BACKGROUND: Among Influenza neuraminidase inhibitors (NAIs), oseltamivir corresponds to the most widely used agent to treat influenza disease. However since 2001, several cases of resistance to NAIs have been reported for circulating seasonal A(H1N1) Influenza viruses. A direct resistance mechanism may be invoked, involving critical mutations in the viral NA gene that prevent the drug binding to its target. Same phenomenon is reported for adamantanes drugs and mutations in the M2 channel protein gene of Influenza viruses. METHODS: Reverse-Transcription/Restriction Fragment Length Polymorphism (RT-PCR/RFLP) method, phenotypic testing for oseltamivir resistance, and sequencing of NA, HA and M2 genes were used in this study. Phylogenetic analyses were performed using BioEdit and Mega 5 softwares for alignment of sequences and phylogenetic trees building respectively. RESULTS: Using a simple RT-PCR/RFLP method, we found that the 86 seasonal A(H1N1) isolates from 2008 bear the oseltamivir resistance-associated mutation (H274Y) in the NA gene. In contrast all isolates isolated in Senegal in 2007 were sensitive to oseltamivir. These results were first confirmed by finding high IC50 values using a phenotypic testing for oseltamivir resistance, and secondly by sequencing the whole NA gene. Regarding M2 gene, no mutation associated to adamantanes resistance was characterized of the isolates. CONCLUSIONS: The present work provides evidence of circulation of drug-resistant seasonal A(H1N1) viruses during the 2008 influenza season (July to September) in Senegal. The results are in favor of multiple introductions of oseltamivir resistant viruses (ORV) A(H1N1) in Senegal.Phylogenetic analyses of isolates with complete sequences of N1 and HA1 genes showed that they belong to clade 2B and suggest sequential introductions in Africa.

Gag p27-specific B- and T-cell responses in Simian immunodeficiency virus SIVagm-infected African green monkeys.

Nonpathogenic simian immunodeficiency virus SIVagm infection of African green monkeys (AGMs) is characterized by the absence of a robust antibody response against Gag p27. To determine if this is accompanied by a selective loss of T-cell responses to Gag p27, we studied CD4(+) and CD8(+) T-cell responses against Gag p27 and other SIVagm antigens in the peripheral blood and lymph nodes of acutely and chronically infected AGMs. Our data show that AGMs can mount a T-cell response against Gag p27, indicating that the absence of anti-p27 antibodies is not due to the absence of Gag p27-specific T cells.

Plasmacytoid dendritic cell dynamics and alpha interferon production during Simian immunodeficiency virus infection with a nonpathogenic outcome.

We addressed the role of plasmacytoid dendritic cells (PDC) in protection against AIDS in nonpathogenic simian immunodeficiency virus (SIVagm) infection in African green monkeys (AGMs). PDC were monitored in blood and lymph nodes (LNs) starting from day 1 postinfection. We observed significant declines in blood during acute infection. However, PDC then returned to normal levels, and chronically infected AGMs showed no decrease of PDC in blood. There was a significant increase of PDC in LNs during acute infection. Blood PDC displayed only weak alpha interferon (IFN-alpha) responses to TLR9 agonist stimulation before infection. However, during acute infection, both blood and LN PDC showed a transiently increased propensity for IFN-alpha production. Bioactive IFN-alpha was detected in plasma concomitant with the peak of viremia, though levels were only low to moderate in some animals. Plasma interleukin 6 (IL-6) and IL-12 were not increased. In conclusion, PDC were recruited to the LNs and displayed increased IFN-alpha production during acute infection. However, increases in IFN-alpha were transient. Together with the lack of inflammatory cytokine responses, these events might play an important role in the low level of T-cell activation which is associated with protection against AIDS in nonpathogenic SIVagm infection.

Antiinflammatory profiles during primary SIV infection in African green monkeys are associated with protection against AIDS.

T cell activation levels in HIV infection are predictive of AIDS progression. We searched for the immunological correlates of protection against disease progression by studying the early stages of nonpathogenic SIV infection in African green monkeys (SIVagm). The African green monkeys (AGMs) displayed high peak viremias and a transient decline in levels of blood CD4(+) and CD8(+) T cells between days 5 and 17 after infection. A concomitant increase in levels of CD4(+)DR(+), CD8(+)DR(+), and CD8(+)CD28(-) cells was detected. After the third week, T cell activation returned to baseline levels, which suggested a protective downregulation of T cell activation. A very early (24 hours after infection) and strong induction of TGF-beta1 and FoxP3 expression was detected and correlated with increases in levels of CD4(+)CD25(+) and CD8(+)CD25(+) T cells. This was followed by a significant increase in levels of IL-10, whereas IFN-gamma gene upregulation was more transient, and levels of TNF-alpha and MIP-1alpha/beta transcripts did not increase in either blood or tissues. The profiles were significantly different during primary SIV infection in macaques (SIVmac); that is, there was a delayed increase in IL-10 levels accompanied by moderate and persistent increases in TGF-beta levels. Together, our data show that SIVagm infection is associated with an immediate antiinflammatory environment and suggest that TGF-beta may participate in the generation of Tregs, which may prevent an aberrant chronic T cell hyperactivation.

Role of monkeys in the sylvatic cycle of chikungunya virus in Senegal.

Arboviruses spillover into humans either as a one-step jump from a reservoir host species into humans or as a two-step jump from the reservoir to an amplification host species and thence to humans. Little is known about arbovirus transmission dynamics in reservoir and amplification hosts. Here we elucidate the role of monkeys in the sylvatic, enzootic cycle of chikungunya virus (CHIKV) in the region around Kédougou, Senegal. Over 3 years, 737 monkeys were captured, aged using anthropometry and dentition, and tested for exposure to CHIKV by detection of neutralizing antibodies. Infant monkeys were positive for CHIKV even when the virus was not detected in a concurrent survey of mosquitoes and when population immunity was too high for monkeys alone to support continuous transmission. We conclude that monkeys in this region serve as amplification hosts of CHIKV. Additional efforts are needed to identify other hosts capable of supporting continuous circulation.

Chikungunya Outbreak in Kedougou, Southeastern Senegal in 2009-2010.

BACKGROUND: In Senegal, Chikungunya virus (CHIKV), which is an emerging mosquito-borne alphavirus, circulates in a sylvatic and urban/domestic cycle and has caused sporadic human cases and epidemics since 1960s. However, the real impact of the CHIKV sylvatic cycle in humans and mechanisms underlying its emergence still remains unknown. METHODOLOGY: One thousand four hundred nine suspect cases of CHIKV infection, recruited from 5 health facilities located in Kedougou region, south-eastern Senegal, between May 2009 to March 2010, together with 866 serum samples collected from schoolchildren from 4 elementary schools in May and November 2009 from Kedougou were screened for anti-CHIKV immunoglobulin (Ig)M antibodies and, when appropriate, for viral nucleic acid by real-time polymerase chain reaction (rPCR) and virus isolation. In addition, mosquitoes collected in the same area from May 2009 to January 2010 were tested for CHIKV by rPCR and by virus isolation, and 116 monkeys sera collected from March 2010 to May 2010 were tested for anti-CHIKV IgM and neutralizing antibodies. RESULTS: The main clinical manifestations of the CHIKV suspect cases were headache, myalgia, and arthralgia. Evidence for CHIKV infection was observed in 1.4% (20 of 1409) of patients among suspect cases. No significant difference was observed among age or sex groups. In addition, 25 (2.9%) students had evidence of CHIKV infection in November 2009. Chikungunya virus was detected in 42 pools of mosquitoes, mainly from Aedes furcifer, and 83% of monkeys sampled were seropositive. CONCLUSIONS: Our findings further documented that CHIKV is maintained in a sylvatic transmission cycle among monkeys and Aedes mosquitoes in Kedougou, and humans become infected by exposure to the virus in the forest.

Phenotype and function of myeloid dendritic cells derived from African green monkey blood monocytes.

Myeloid dendritic cells probably play an important role in the immune response against HIV and SIV, and in the enhancement of CD4+ T cell infection. Here, we have investigated phenotypic and functional features of myeloid monocyte-derived DC (MDDC) from African green monkeys (AGMs). AGMs are natural hosts of SIV and exhibit no signs of abnormal T cell activation despite high SIV plasma viremia. We identified mAbs that cross-react specifically with homologous molecules expressed on AGM DC. We adapted a protocol to derive AGM MDDC by culture in the presence of GM-CSF and IL-4. The differentiated cells possessed a typical dendritic morphology and the majority were CD11c+ DC-SIGN+. AGM MDDC displayed a high expression of typical maturation markers, such as CD83, CD86 and DC-LAMP, and moderate immunostimulatory capacity, suggesting that the cells were in a semi-mature state. Stimulation resulted in further maturation, as shown by up-regulation of CD80 and decrease of endocytosis ability. However, neither increase of HLA-DR or CD40 expression nor enhanced immunostimulatory capacity was observed. The latter was associated with a low pro-inflammatory cytokine production during mixed lymphocyte reactions and a cytokine balance in favour of IL-10 in contrast to human MDDC. This is the first characterization of AGM MDDC. The tools described here are a crucial step for future studies in vivo or in vitro on the function of myeloid DC using the AGM animal model.

Impact of viral factors on very early in vivo replication profiles in simian immunodeficiency virus SIVagm-infected African green monkeys.

To better understand which factors govern the levels of viral loads in early lentiviral infections of primates, we developed a model that allows distinguishing between the influences of host and viral factors on viremia. Herein we report that two species of African green monkeys (Chlorocebus sabaeus and C. pygerythrus) infected with their respective wild-type simian immunodeficiency virus SIVagm viruses (SIVagm.sab92018 and SIVagm.ver644) consistently showed reproducible differences in viremia during primary infection but not at later stages of infection. Cross-infections of SIVagm.sab92018 and SIVagm.ver644 into, respectively, C. pygerythrus and C. sabaeus revealed that the dynamics of viral replication during primary infection were dependent on the viral strain used for the infection but not on the host. Hence, the kinetics of SIVagm.sab92018 and SIVagm.ver644 were similar in both sabaeus and vervet animals, indicating that the difference in viremia levels between the two groups during the early phase of infection was not associated with the host. Coreceptor usage for these two strains showed a larger coreceptor repertoire for SIVagm.sab92018, which is able to efficiently use CXCR4 in addition to CCR5, than for SIVagm.ver644, which showed a classical CCR5 coreceptor usage pattern. These differences could not be explained by different charges of the V3 loop for SIVagm.sab92018 and for SIVagm.ver644. In conclusion, our study showed that the extent of virus replication during the primary infection is primarily dependent on viral determinants.

DC-SIGN from African green monkeys is expressed in lymph nodes and mediates infection in trans of simian immunodeficiency virus SIVagm.

African green monkeys (AGMs) infected by simian immunodeficiency virus (SIV) SIVagm are resistant to AIDS. SIVagm-infected AGMs exhibit levels of viremia similar to those described during pathogenic human immunodeficiency virus type 1 (HIV-1) and SIVmac infections in humans and macaques, respectively, but contain lower viral loads in their lymph nodes. We addressed the potential role of dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN; CD209) in viral dissemination. In previous studies, it has been shown that human DC-SIGN and macaque DC-SIGN allow transmission of HIV and SIVmac to T cells. Here, we looked at the ability of DC-SIGN derived from AGM lymph nodes to interact with SIVagm. We show that DC-SIGN-expressing cells are present mainly in the medulla and often within the cortex and/or paracortex of AGM lymph nodes. We describe the isolation and characterization of at least three isoforms of dc-sign mRNA in lymph nodes of AGMs. The predicted amino acid sequence from the predominant mRNA isoform, DC-SIGNagm1, is 92 and 99% identical to the corresponding human and rhesus macaque DC-SIGN amino acid sequences, respectively. DC-SIGNagm1 is characterized by the lack of the fourth motif in the repeat domain. This deletion was also detected in the dc-sign gene derived from thirteen animals belonging to five other African monkey species and from four macaques (Macaca fascicularis and M. mulatta). Despite three- to seven-amino-acid modifications compared to DC-SIGNmac, DC-SIGNagm1 allows transmission of SIVagm to T cells. Furthermore, AGM monocyte-derived dendritic cells (MDDC) expressed at least 100,000 DC-SIGN molecules and were able to transmit SIVagm to T cells. At a low multiplicity of infection (10(-5) 50% tissue culture infective doses/cell), viral transmission by AGM MDDC was mainly DC-SIGN dependent. The present study reveals that DC-SIGN from a natural host species of SIV has the ability to act as an efficient attachment and transmission factor for SIVagm and suggests the absence of a direct link between this ability and viral load levels in lymph nodes.