Pandemic influenza A(H1N1) 2009: molecular characterisation and duration of viral shedding in intensive care patients in Bordeaux, south-west France, May 2009 to January 2010.

From May 2009 to January 2010, the Virology Laboratory at the University Hospital of Bordeaux received more than 4,000 nasopharyngeal samples from the Aquitaine region (south-west France) for the diagnosis of pandemic influenza A(H1N1)2009. Eighty-three infected patients deteriorated and were admitted to intensive care units. Our study focused on 24 of these patients. Positivity for influenza A(H1N1)2009 was monitored by realtime PCR and duration of viral shedding was determined. The first available sample of each patient was analysed for bacterial, fungal and viral co-infection. We observed six bacterial (or bacterial/fungal) co-infections and one viral co-infection with respiratory syncytial virus. The samples were analysed for the presence of the neuraminidase H275Y (N1 numbering) mutation, which confers resistance to oseltamivir, by realtime PCR of the neuraminidase gene. No H275Y mutation was observed in any of the viral strains screened in this study. In parallel, a fragment of the haemagglutinin gene encoding amino acid residues 173 to 362 was sequenced to detect mutations that had been reported to increase the severity of the disease. Two patients were infected by strains bearing the D222G (H3 numbering) mutation. The viral shedding of A(H1N1)2009 in this study ranged from four to 28 days with a median of 11 days.

Human polyomavirus JC latency and reactivation status in blood of HIV-1-positive immunocompromised patients with and without progressive multifocal leukoencephalopathy.

BACKGROUND: Human polyomavirus JC (JCV) induces human progressive multifocal leukoencephalopathy (PML) in patients with AIDS. Peripheral blood mononuclear cells (PBMC) of HIV-1-positive immunocompetent and immunocompromised patients can harbour JCV genome, but their precise role in JCV latency or reactivation status before the onset of PML remains hypothetical. OBJECTIVES: To assess JCV latency or reactivation status in PBMC of HIV-1-positive immunocompromised patients without PML. DESIGN: A group of 82 HIV-1-positive immunocompromised patients who did not have PML were compared with 10 patients with AIDS and PML and with 69 HIV-1-positive immunocompetent patients without PML. METHODS: DNA and total RNA were extracted from PBMC. The presence of JCV DNA was demonstrated by a semi-nested polymerase chain reaction (PCR). By using primer pairs specific for an early gene,T, and a late gene, VP1, the expression of both early and late gene mRNA in PBMC could be identified using reverse transcriptase (RT) PCR. RESULTS: JCV DNA was detected by PCR in 17.4% of 69 HIV-1-positive immunocompetent patients, in 23.2% of 82 HIV-1-positive immunocompromised patients, and in 60% of 10 patients with AIDS and PML. No correlation could be drawn between the detection of JCV DNA in the PBMC and the clinical or biological status of the HIV-1-positive patients. By using RT-PCR procedures, no expression of JCV early and late mRNA in PBMC was found in any patients. CONCLUSIONS: JCV DNA is detectable in the PBMC of 20.5% of 151 HIV-1-infected patients independently of the CDC (Centers for Disease Control and Prevention) stages of the infection. Moreover, our results suggest that active replication of JCV in PBMC appears to be absent or at least a very rare event in HIV-1-positive immunocompromised patients with and without PML.

Detection of JC virus DNA in the peripheral blood leukocytes of HIV-infected patients.

OBJECTIVES: To assess whether JC virus (JCV) DNA is frequently harboured by peripheral blood leukocytes (PBL) in HIV-positive patients, before the onset of progressive multifocal leukoencephalopathy (PML). DESIGN: The polyomavirus JCV induces PML in immunocompromised persons and particularly AIDS patients. Leukocytes may play a central part in the onset of PML, but their precise role in JCV latency and reactivation still remains hypothetical. The controversial presence of JCV DNA in PBL has been, until now, investigated only among small groups of patients. We therefore studied 157 HIV-positive persons and compared them with 65 HIV-negative immunocompromised patients. METHODS: DNA was extracted from PBL. The presence of JCV DNA was demonstrated by the polymerase chain reaction (PCR) alone or combined with a molecular hybridization assay. RESULTS. The presence of JCV DNA was ascertained by PCR and hybridization in 28.9% of 135 HIV-infected persons at all stages of HIV infection and only 16.4% of 61 HIV-negative immunocompromised patients. No correlation could be drawn between the detection of JCV DNA and the clinical or biological status of the HIV-positive patients. CONCLUSIONS: JCV DNA is detectable in the PBL of 28.9% of HIV-infected persons, even in the early stages of infection. JCV is more seldomly amplified in HIV-negative immunocompromised patients. Further work is in progress to determine the prognostic value of the presence of JCV DNA in the blood of HIV-positive patients.

In vitro assays show a dissociation of reverse transcriptase activity and core antigen (p24) production in two HIV-1 isolates from a patient receiving long-term treatment with zidovudine (ZDV).

Two HIV-1 isolates were obtained from a patient receiving long-term treatment with zidovudine (ZDV). The in vitro sensitivity to ZDV triphosphate of the reverse transcriptase (RT) from both isolates appeared to be unchanged compared to that of the LAV-Bru HIV-1 reference strain. When isolates were grown in CEM cells (a T-lymphoblastoid tumor cell line) and their RT activity and core antigen (p24) production were determined, the level of p24 production compared to RT activity was high; in infected CEM cells treated with ZDV, RT activity was at background level while the p24 production was still significant, thus indicating a dissociation of RT activity and core antigen production.

Zidovudine resensitization and dual HIV-1 resistance to zidovudine and lamivudine in the delta lamivudine roll-over study.

OBJECTIVE: To study zidovudine resensitization and dual resistance to zidovudine/lamivudine in HIV-1 isolates from nucleoside reverse transcriptase (RT) inhibitor-experienced patients during selective pressure exerted by zidovudine/lamivudine combination therapy. DESIGN AND METHODS: HIV-1 isolates from 29 patients receiving zidovudine/lamivudine combination therapy in the Delta roll-over study were analysed at entry and during a 1 year follow-up period for phenotypic susceptibility to zidovudine and lamivudine in the ANRS PBMC assay. The RT gene from codon 20 to 230 and at codon 333 was analysed by nucleotide sequencing of the corresponding isolates. RESULTS: HIV-1 isolates from 23 of the 29 patients were phenotypically resistant to zidovudine at baseline; 61% of these patients showed significant zidovudine resensitization during follow-up. The zidovudine IC50 value correlated positively with log10 plasma HIV-1 RNA (P = 0.02) and negatively with the CD4 cell count (P = 0.004). Zidovudine resensitization (related to acquisition of the M184V mutation) was transient, with evolution towards dual resistance to zidovudine and lamivudine in 20 of the 29 patients. The phenotype of certain dually resistant isolates coincided with the emergence of multiple mutations in the 5' part of the RT gene. CONCLUSIONS: M184V-mediated zidovudine resensitization of HIV-1 is transient in most patients who are given zidovudine/lamivudine combination therapy when zidovudine resistance has already emerged. The subsequent evolution towards dual phenotypic resistance to zidovudine/lamivudine corresponds to complex genotypic profiles.

Course of specific T lymphocyte cytotoxicity, plasma and cellular viral loads, and neutralizing antibody titers in 17 recently seroconverted HIV type 1-infected patients.

Relationships were sought between specific anti-HIV cytotoxic T lymphocyte (CTL) responses (against structural and regulatory proteins of the HIV-1 LAI isolate) and plasma and cellular viral loads (VLs) in 17 recently HIV-1-infected patients including 3 displaying asymptomatic primary infection (PI) followed up for 12 months. Plasma VL was correlated directly with CD8 counts and inversely with CD4 counts. Cytotoxic reactions were observed in all patients and directed mainly against structural proteins. The earliest CTL responses were against Gag and Env proteins detected in 87 and 75% of the subjects, respectively, within the first month following PI. Anti-Env and Gag cytotoxic responses were inversely correlated with the plasma VL. Reactions against the pol gene products were thought to be either less involved in or less efficient for the initial decrease of viremia. Responses against regulatory gene products were weak and variable, apart from Nef, which was recognized by half of the subjects. Neutralizing antibodies were not detected before month 3, and were found only in six patients at subsequent times. Two of three patients with asymptomatic PI had a low viral burden and either a delayed response or one limited to a few protein CTL responses, suggesting that the magnitude of the CTL response depends on the initial plasma VL. The third patient displayed viral and CTL parameters identical to those of the patients with symptomatic PI. However, two subjects with symptomatic PI exhibited similarly low plasma VL and moderate CTL responses. Overall, the results suggest that the CTL response may not be the sole factor controlling viremia.

HIV type 1 Thai subtype E is predominant in South Vietnam.

PIP: Samples of peripheral blood mononuclear cells from 50 HIV-1-infected individuals in South Vietnam were analyzed to determine with which HIV-1 subtype the subjects were infected. Participants were from Ho Chi Minh city and five surrounding provinces; 16 samples from female prostitutes, 32 from IV drug users, and one each from a man and woman not in any HIV risk group. 32 individuals were therefore most likely infected by IV drug use and the rest through sexual contacts. PCR amplification and heteroduplex mobility assay found all but one case to be infected with HIV-1 subtype E. The only nonsubtype E infection was HIV-1 subtype B in a woman sexually infected by her seropositive partner who was most likely exposed to the virus in Europe. HIV-1 subtype E strongly predominates in South Vietnam. The homogeneous geographic distribution of subtype E suggests the recent introduction of the virus into the country. A Thai origin can be considered given the genetic relationship between the Thai and Vietnamese subtypes E. It may be assumed that subtype E infections of Vietnamese prostitutes are related to the progressive entry and spread of HIV-1 subtype E from Thailand to Cambodia and then to southern Vietnam.^ieng

Sequence analysis of env C2/V3, gag p17/p24, and pol protease regions of 25 HIV type 1 isolates from Ho Chi Minh City, Vietnam.

Env C2/V3, gag p17/p24, pol protease, and RT regions of HIV-1 isolates recently obtained from 25 HIV-1 seropositive individuals from Ho Chi Minh City (Vietnam) were studied, and genes subtypes were determined by DNA sequence analyses. Twenty-three isolates out of 25 were identified as belonging to subtype E, now recognized as circulating recombinant form 1 (CRF01_AE). The motif at the top of the V3 loop (generally GPGQ) was then preceded by an isoleucine or a methionine (M) residue; the M residue might be a local signature of Vietnamese E isolates compared to Thai E viruses. Two isolates (8%) were shown to be intersubtype recombinants: one E/B and one CRF02_AG(IBNG)/D. The polymorphism of pol protease was considered only for CRF01_AE isolates and is clearly different from that recorded for B viruses with substitutions at positions 13, 35, 36, 41, 69, and 89.

PCR detection of human enteric viruses in bathing areas, waste waters and human stools in Southwestern France.

Detection of human pathogenic viruses by molecular techniques might be suitable for identifying viral pollution in environmental waters and for improving diagnosis in patients. Environmental samples were taken from bathing areas and sewage treatment plants in southwestern France. Small volume samples (50 microL) were tested. Five groups of enteric pathogenic viruses were studied: enteroviruses, Norwalk-like viruses (NLVs), hepatitis A virus, rotaviruses and adenoviruses. Moreover, human samples were tested for NLV. After extraction of viral nucleic acids (Boom's procedure), a nested polymerase chain reaction was conducted before hybridization. Five bathing waters out of 26 were positive for one viral group, without systematic association with bacterial contamination. Eight sewage plant samples out of 13 were positive for at least one viral group. Seven patients out of 45 were NLV-positive. Molecular techniques allow efficient screening of viral contamination in environmental waters and the study of NLV molecular epidemiology.

Qualitative and quantitative molecular detection of enteroviruses in water from bathing areas and from a sewage treatment plant.

Pathogenic enteric viruses can be introduced into the environment as a result of human activities. Enteroviruses are regularly detected in environmental waters or shellfish and can provoke potentially serious diseases. Some authors believe that enteroviruses could represent an interesting indicator of viral contamination in the environment. Since molecular approaches seem to be promising for the detection of these viruses, we developed a simple qualitative RT-PCR procedure for enteroviruses, together with a quantitative RT-PCR assay using RNA internal standard. After one-tube-RT-PCR, this standard and wild enterovirus RNA were detected by differential hybridization with specific probes and a fluorimetric reaction. The quantification of enteroviruses, conducted in a sewage treatment plant, showed a decreasing number of genomic copies from the entrance to the exit (from 3.8 x 10(5) to 5.4 x 10(4) RNA copies/mL) but indicated the presence of enterovirus RNA in the neighboring river (2.2 x 10(3) RNA copies/mL). In bathing areas, enterovirus RNA was detected in 16 out of 226 samples, with copies numbers ranging from 3.7 x 10(2) RNA copies/mL to 7 x 10(4) RNA copies/mL.

Serological evidence of human infection with the paramyxovirus Yucaipa in Senegal, West Africa.

Sera from 353 people resident in Dakar, Senegal, West Africa, were tested for hemagglutination-inhibiting antibodies against the avian paramyxovirus Yucaipa. Antibodies were demonstrated in five sera (1.4%), providing new evidence that this virus may infect humans.

Clinical and biological characteristics of human immunodeficiency virus-infected and uninfected intravascular drug users in Ho Chi Minh City, Vietnam.

To define the medical characteristics of intravascular drug users in Ho Chi Minh City, Vietnam, we examined 280 men, of whom 235 were infected with human immunodeficiency virus (HIV), being treated in a rehabilitation center. The patients used mainly opium, often in shooting galleries (50%). The prevalence of oral candidiasis (58%) and zoster infection (20%) was high in HIV-seropositive patients, whereas oral hairy leukoplasia and Kaposi's sarcoma were absent. The prevalence of acquired immunodeficiency syndrome was 24%. More than 80% of the patients had infections with hepatitis C virus, hepatitis B virus, cytomegalovirus, or human T cell lymphotropic virus type-1. The CD4+ cell counts correlated well with viral load. Only HIV-1 subtype E was detected in the 30 patients tested. A cohort study of HIV-infected subjects in this population seems feasible, and would permit introduction of anti-retroviral therapy The large number of HIV-seronegative subjects sharing the same at-risk practices as the HIV-infected subjects raises the possibility of natural protection in this population.

Detection of the bunyavirus Germiston in VERO and Aedes albopictus C6/36 cells by in situ hybridization using cDNA and asymmetric RNA probes.

Using Germiston virus infected vertebrate (VERO) and invertebrate (Aedes albopictus C6/36) cells, paraformaldehyde-glutaraldehyde fixative allowed the best preservation of cellular morphology and the highest hybridization signals with cDNA and asymmetric RNA probes against the viral S segment. Asymmetric RNA probes always gave higher sensitivity and better specificity of in situ hybridization than the nick-translated symmetric DNA probe in both vertebrate and invertebrate cells. The study of Aedes albopictus C6/36 cells persistently infected with Germiston virus showed that only a small number of cells contained the S segment, and that the replication and transcription of the S segment took place in the cytoplasm of acutely and persistently infected cells.

A hospital outbreak of gastroenteritis possibly related to the contamination of tap water by a small round structured virus.

Small round structured viruses (SRSVs) are a major cause of gastroenteritis in institutions and sensitive new molecular techniques allow rapid diagnosis and the establishment of control measures. In January 1999, a 10 day-long outbreak of gastroenteritis in a re-education ward, was reported by a hospital hygiene department. A potential common source of contamination was tap water. The stools of six patients with gastroenteritis and seven tap water samples from the hospital ward, were tested for SRSV by reverse transcription and polymerase chain reaction (RT-PCR): three stools and four water samples, all bacteriologically negative, were SRSV-positive. Nucleotide sequencing of a fragment of the SRSV polymerase gene showed that the sequences of the positive samples (two patients and four water samples) were identical (genogroup II). We cannot exclude interhuman transmission of SRSV together with viral soiling of some taps in the ward, but this hospital infection was more likely due to the transient contamination of the ward supply of drinking water with a SRSV strain.

Hepatitis B exacerbation with a precore mutant virus following withdrawal of lamivudine in a human immunodeficiency virus-infected patient.

Chronic active hepatitis B exacerbations have been reported following development of resistance to or withdrawal of lamivudine in HIV-infected patients. A 38-year-old woman with HIV and chronic HBV infections was hospitalized because of acute hepatitis. The occurrence of cytolysis with replication of HBV 2 months after withdrawing lamivudine suggests that our patient experienced a severe reactivation of HBV infection due to the modification of her treatment. Sequencing of the HBV precore region showed the strain to be a mutant. We conclude that lamivudine should not be stopped in HIV- and HBV-infected patients, but could be continued at the dose of 100mg/day as used in isolated HBV infection.

First isolation of a Yucaipa-like virus in Africa.

A virus having a hemagglutinin closely related to that of paramyxovirus Yucaipa was isolated from the feces of a wild bird in Senegal, West Africa.

A highly virulent togavirus-like agent associated with the fulminating disease of guinea fowl.

During a 1986 natural lethal outbreak of fulminating disease in guinea poult flocks in southwestern France, enveloped virus particles were consistently observed in the gut contents of infected birds. For the present study, a protocol was developed for the purification of these particles. Sucrose-banded virus obtained from birds infected experimentally with virus from the outbreak was found to have a buoyant density of 1.18 g/ml. The purified virus showed hemagglutinating activity, was shown by electron microscopy to have a togavirus-like morphology, and also was shown to be transmissible and pathogenic through oral ingestion. In addition, other enveloped particles have been occasionally detected in gut contents of both infected and uninfected birds; the improbability of the viral nature of these interfering particles is discussed.

Experimental infection of Columba livia with paramyxovirus Yucaipa.

The town pigeons (Columba livia) were inoculated intranasally with a Yucaipa-like virus (PLOC/Senegal/273/77). They were then surveyed for virus production in the cloaca over a 10 day period. The virus could be isolated at two, three and four days after inoculation.

[Comparative study of 2 commercial tests for the detection of HIV-1 antigens in blood and cerebrospinal fluid using immunoenzymology]. / Etude comparative de deux trousses commerciales pour la recherche de l'antigène HIV I en immunoenzymologie (EIA) dans le sérum et le liquide céphalorachidien.

The authors have carried out, on 150 sera of patients seropositive for the human immunodeficiency virus type I (HIV I) and 11 cerebrospinal fluid of which 5 were patient infected by the HIV I, a comparative study of two commercial tests for the detection of HIV I antigen (Diagnostic Pasteur and Abbott laboratories). A much greater sensitivity was obtained with the specificity being practically identical for the sera with the two tests (100% with Abbott laboratories test, 96.11% with the diagnostic Pasteur test). 4 sera appeared "false negatives" with the Abbott Laboratories test; their optical density was situated between 80 and 100 p. cent of the cut-off level value, whereas that of the "real" negatives was situated between 30 and 60 p. cent of the cut-off level value. 10 of the 11 cerebrospinal fluids appeared false positive with the Diagnostic Pasteur. This seems to be connected with an insufficiency of saturation of protein receptors in the wells. The Diagnostic Pasteur test is not adapted for the detection of HIV I antigen in the body fluids with a weak protein concentration. Contrary to the results obtained with the Encavor test (Abbott laboratories) the analysis in western-blot does not show an inverse prevalence of anti p24 GAG antibodies with regard to antigen HIV I in seropositive patients. On the other hand, the statistical analysis of the positive HIV I sera which are at the same time antigen HIV I positive and antibodies HIV I positive suggests an earlier disappearance of anti p17 GAG antibodies than of anti p24 GAG antibodies.

[The inhibiting effect of anticholera vaccination carried out simultaneously or at short intervals on yellow fever immunization. Is it real or assumed? Results of a retrospective study]. / L'effet inhibiteur de la vaccination anticholérique simultanée ou rapprochée sur l'immunisation contre la fièvre jaune est-il réel ou supposé? Résultats d'une étude rétrospective.

121 serum samples from African adults previously immunised with 17 D yellow fever vaccine alone (control group) or simultaneous yellow fever and cholera vaccines were tested for yellow fever antibodies by seroneutralization and haemagglutination inhibition assays. Comparison of the rates of seroconversion and antibody titers between the groups vaccinated the same day or into a short (less than or equal to 10 days) or a long time interval with both vaccines and the control group gave no significant difference. The association of cholera and yellow fever vaccines do not influence the long-term efficiency of yellow fever vaccination.