Need for integrative thinking to fight against emerging infectious diseases. Proceedings of the 5th seminar on emerging infectious diseases, March 22, 2016 - current trends and proposals.

We present here the proceedings of the 5th seminar on emerging infectious diseases, held in Paris on March 22nd, 2016, with seven priority proposals that can be outlined as follows: encourage research on the prediction, screening and early detection of new risks of infection; develop research and surveillance concerning transmission of pathogens between animals and humans, with their reinforcement in particular in intertropical areas ("hot-spots") via public support; pursue aid development and support in these areas of prevention and training for local health personnel, and foster risk awareness in the population; ensure adapted patient care in order to promote adherence to treatment and to epidemic propagation reduction measures; develop greater awareness and better education among politicians and healthcare providers, in order to ensure more adapted response to new types of crises; modify the logic of governance, drawing from all available modes of communication and incorporating new information-sharing tools; develop economic research on the fight against emerging infectious diseases, taking into account specific driving factors in order to create a balance between preventive and curative approaches.

First cases of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) infections in France, investigations and implications for the prevention of human-to-human transmission, France, May 2013.

In May 2013, Middle East Respiratory Syndrome Coronavirus (MERS-CoV) infection was diagnosed in an adult male in France with severe respiratory illness, who had travelled to the United Arab Emirates before symptom onset. Contact tracing identified a secondary case in a patient hospitalised in the same hospital room. No other cases of MERS-CoV infection were identified among the index case's 123 contacts, nor among 39 contacts of the secondary case, during the 10-day follow-up period.

Investigation of a mpox outbreak in Central African Republic, 2021-2022.

Human monkeypox virus is spreading globally, and more information is required about its epidemiological and clinical disease characteristics in endemic countries. We report the investigation of an outbreak in November 2021 in Central African Republic (CAR). The primary case, a hunter, fell ill after contact with a non-human primate at the frontier between forest and savannah. The ensuing investigation in a small nearby town concerned two families and four waves of inter-human transmission, with 14 confirmed cases, 11 suspected cases and 17 non-infected contacts, and a secondary attack rate of 59.5% (25/42). Complications were observed in 12 of the 19 (63.2%) confirmed and suspected cases with available clinical follow-up data: eight cases of bronchopneumonia, two of severe dehydration, one corneal ulcer, one abscess, two cutaneous superinfections, and six cutaneous sequelae (cheloid scars, or depigmentation). There was one death, giving a case fatality ratio of 1/25 (4.0%) for confirmed and suspected cases. This outbreak, with the largest number of confirmed cases ever described in CAR, confirms the potential severity of the disease associated with clade I monkeypox viruses, and highlights the need for rapid control over virus circulation to prevent the further national and international spread of infection.

Impact of trace elements and vitamin supplementation on immunity and infections in institutionalized elderly patients: a randomized controlled trial. MIN. VIT. AOX. geriatric network.

BACKGROUND: Antioxidant supplementation is thought to improve immunity and thereby reduce infectious morbidity. However, few large trials in elderly people have been conducted that include end points for clinical variables. OBJECTIVE: To determine the effects of long-term daily supplementation with trace elements (zinc sulfate and selenium sulfide) or vitamins (beta carotene, ascorbic acid, and vitamin E) on immunity and the incidence of infections in institutionalized elderly people. METHODS: This randomized, double-blind, placebo-controlled intervention study included 725 institutionalized elderly patients (>65 years) from 25 geriatric centers in France. Patients received an oral daily supplement of nutritional doses of trace elements (zinc and selenium sulfide) or vitamins (beta carotene, ascorbic acid, and vitamin E) or a placebo within a 2 x 2 factorial design for 2 years. MAIN OUTCOME MEASURES: Delayed-type hypersensitivity skin response, humoral response to influenza vaccine, and infectious morbidity and mortality. RESULTS: Correction of specific nutrient deficiencies was observed after 6 months of supplementation and was maintained for the first year, during which there was no effect of any treatment on delayed-type hypersensitivity skin response. Antibody titers after influenza vaccine were higher in groups that received trace elements alone or associated with vitamins, whereas the vitamin group had significantly lower antibody titers (P<.05). The number of patients without respiratory tract infections during the study was higher in groups that received trace elements (P = .06). Supplementation with neither trace elements nor vitamins significantly reduced the incidence of urogenital infections. Survival analysis for the 2 years did not show any differences between the 4 groups. CONCLUSIONS: Low-dose supplementation of zinc and selenium provides significant improvement in elderly patients by increasing the humoral response after vaccination and could have considerable public health importance by reducing morbidity from respiratory tract infections.

Increased influenza A virus sialidase activity with N-acetyl-9-O-acetylneuraminic acid-containing substrates resulting from influenza C virus O-acetylesterase action.

Influenza virus type C (Johannesburg/1/66) was used as a source for the enzyme O-acetylesterase (EC 3.1.1.53) with several natural sialoglycoconjugates as substrates. The resulting products were immediately employed as substrates using influenza virus type A [(Singapore/6/86) (H1N1) or Shanghai/11/87 (H3N2)] as a source for sialidase (neuraminidase, EC 3.2.1.18). A significant increase in the percentage of sialic acid released was found when the O-acetyl group was cleaved by O-acetylesterase activity from certain substrates (bovine submandibular gland mucin, rat serum glycoproteins, human saliva glycoproteins, mouse erythrocyte stroma, chick embryonic brain gangliosides and bovine brain gangliosides). A common feature of all these substrates is that they contain N-acetyl-9-O-acetylneuraminic acid residues. By contrast, no significant increase in the release of sialic acid was detected when certain other substrates could not be de-O-acetylated by the action of influenza C esterase, either because they lacked O-acetylsialic acid (human glycophorin A, alpha 1-acid glycoprotein from human serum, fetuin and porcine submandibular gland mucin) or because the 4-O-acetyl group was scarcely cleaved by the viral O-acetylesterase (equine submandibular gland mucin). The biological significance of these facts is discussed, relative to the infective capacity of influenza C virus.

Genic amplification of the entire coding region of the HEF RNA segment of influenza C virus.

In order to provide an easy and powerful analysis of influenza C viral HEF RNA segment of a recent strain, a combination of reverse transcription and the polymerase chain reaction was used. We amplified the entire coding region of the HEF gene of a laboratory strain of virus called C/Johannesburg/1/66, widely used for binding and esterase activity studies as well as that of a strain isolated in 1991 (C/Paris/145/91) from a patient suffering from severe flu syndrome. The sequences we amplified were about 2 kilobases long. In this work, we show that the forward 'universal primer' Uni1, which has been used for influenza A and B viruses cDNA syntheses can also be used for influenza C virus. The PCR primers were designed to contain restriction sites to make the PCR products ready to be used for further purposes. A restriction analysis of the PCR products combined with analyses of all the human influenza C virus HEF gene sequences published so far permitted the design of sets of oligonucleotides which can prime PCR on cDNA of unknown influenza C virus for cloning.

A nylon membrane enzyme immunoassay for rapid diagnosis of influenza A infection.

A new membrane-enzyme immunofiltration assay (MIFA) was developed for rapid diagnosis of influenza A infection. The pretreated specimens were dispensed into a 1.2 micron Biodyne B nylon membrane-bottomed microplate and vacuum filtration was applied. Blocking solution, peroxidase-conjugated anti-influenza A nucleoprotein monoclonal antibody, washing buffer and substrate were added in that order. The assay was completed within 30 min. Out of 103 nasopharyngeal swabs collected in transport medium, 31 isolates of influenza A virus were obtained and 22 specimens were detected directly by the MIFA technique. The 9 isolation-positive MIFA-negative specimens required 6 days or more for viral detection in cell culture, and probably contained a very low quantity of virus. The 72 cell culture negative specimens were also negative by MIFA. Comparison with a classical immunocapture assay (ICA) gave a better sensitivity for MIFA, as only 15/103 specimens were positive by ICA. MIFA is a rapid test with 71% sensitivity and 100% specificity. It was also very useful to test the cell culture supernatants, as a sensitivity of 100% was obtained with MIFA when the immunofluorescence technique was positive. The same technique could be readily carried out on the same plate for other respiratory viruses since capture antibody is not used.

Influenza C virus infection in France.

Little is known of the epidemiology of influenza C virus infections in western Europe and of the exact role of this agent in acute viral respiratory infections. Several tests may be used for detecting antibodies against this agent but the significance of their respective results is not clear. A total of 301 samples of serum was collected from persons aged from 4 months to 88 years living in France in 1988. The samples were tested for the presence of antibodies to influenza C virus by haemagglutination-inhibition (HI) tests and ELISA. The specificity of the results was checked by immunoblotting and by antibody absorption with staphylococcal protein A. Significant HI activity was found in 61% of the 301 samples tested, titres ranging from 20-320; 70% were positive by ELISA with titres ranging from 500 to 32,000. The population tested was divided into four age groups: 0-15 years; 16-30 years; 31-50 years and 51-88 years. The highest rates for positive samples were found in the 16-30 year group (76 and 79% by HI tests and ELISA respectively) as well as significant HI and ELISA geometric mean titres. Positive samples were less common in young children (46 and 50% by HI tests and ELISA respectively) and in the oldest group (44 and 54% respectively). The 31-50 years age group formed an intermediate class. The high prevalence of antibody as well as the significant titres indicate intense circulation of influenza C virus, especially among young adults.

Evidence for evolutionary stasis and genetic drift by genetic analysis of two equine influenza H3 viruses isolated in France.

The amino acid sequences of the HA(1) portion of the haemagglutinin of two equine A(H3N8) influenza viruses isolated in France in 1993 and 1998 were analysed to determine their evolutionary relationship with 51 other HA(1) amino acid sequences available in databanks. Our data show that the French strain isolated in 1993 belongs to a group of phylogenetically related viruses branched on the main trunk, illustrating the main lineage of evolution of the equine-2 H3 sequences before its split into two distinct lineages in the late 1980s. By contrast, the 1998 French isolate appears to belong to the more recent 'Eurasian' lineage. These data suggest that equine-2 strains antigenically related to old prototype viruses may cocirculate with the more recent 'Eurasian' and 'American' lineages. In conclusion, it may be necessary to include both strains representative of recent equine influenza variants and an older prototype strain in the current equine influenza vaccines.

European Influenza Surveillance Scheme on the Internet.

In 1995, The European Influenza Surveillance Scheme was created with the participation of eight networks from seven countries. The main objectives were to continue the previous CARE Telematics Network and to adapt the project to the Internet environment as well as to improve substantially the quality of the surveillance according to new epidemiological requirements. Clinical and virological data from the general population and hospitals are collected in an interactive real-time database which can then be used for data entry, queries and consultations. Research programmes have been undertaken in various fields such as standardisation of clinical data and comparability between countries. Validation and security processes guarantee the quality assurance as well as regular assessment by the steering committee. Two additional countries will participate during the next influenza season (1997-98). This will represent an early warning system in a region of approximately 264 million inhabitants.

Production of influenza virus in cell cultures for vaccine preparation.

Influenza virus strains of different types for use as an inactivated vaccine have been successfully grown in different cell lines. Increasing titres were obtained with BHK-21/BRS, VERO and MDCK cells. Cultures in stationary flasks, in spinner cultures or in large bioreactor systems were tested and the optimal conditions were studied. MDCK cells grown in serum-free medium before and during the virus production phase were found to yield high titres in the presence of trypsin. Satisfactory results were obtained with egg-adapted strains of human and equine origin as well as with strains just isolated from human patients without any further passages in eggs or cell culture.

Surveillance of influenza in Europe from October 1999 to February 2000.

Surveillance of influenza through EISS (European Influenza Surveillance Scheme) during the 1999 to 2000 winter shows that influenza affected most of the 11 participating countries and was particularly active in December 1999 and January 2000. Influenza A(

Monitoring of influenza in the EISS European network member countries from October 2000 to April 2001.

In countries covered by the European Influenza Surveillance Scheme (EISS), the 2000-2001 winter was marked mainly by the spread of influenza A(H1N1) viruses. Influenza B, which globally represented a minority of cases, was common later in the season and predominant in Great Britain, Ireland, and Portugal. Influenza activity was at its maximum during the period of January and February/March 2001 with little time lag between countries (maximum four weeks). Overall, the morbidity rates reported were much lower than for the previous season, illustrating a moderate level of influenza activity.

Natural infection of dogs by influenza C virus: a serological survey in Spain.

Two seroepidemiological surveys carried out so far, one in Japan, the other in France, gave a strong indication that dogs may be naturally infected by influenza C virus, considered to be exclusively human until recently. In this work, 101 serum samples were collected during winter 1989/1990 from dogs in Castilla y León, Spain. Sera were tested for the presence of antibodies to influenza C virus by Hemagglutination Inhibition (HI) test. Using antibody absorption by staphylococcal protein A, we demonstrated the specificity of the results. Significant HI activity was found in 56.3% of the 101 tested sera and titres ranged from 25 to 200.

[Epidemiology of SARS: mission of the emergency medical department of the French Hospital of Hanoi]. / Epidémie de pneumopathie atypique: mission SAMU à l'Hôpital Français de Hanoï.

During the epidemic of severe acute respiratory syndrome (SARS) that occurred in Vietnam in March 2003, the French Ministries of Health and Foreign Affairs dispatched a mission composed of personnel from the emergency medical assistance department (French acronym, SAMU) and one virologist from the Pasteur Institute to the French hospital in Hanoi. The purpose of this mission was to reinforce the local medical staff, to bring medical equipment, and to assist in identifying the cause of the SARS epidemic. Most of the 39 cases observed involved health care personnel working at the hospital. Six including 5 who died presented severe manifestations. Application of strict empirical measures of isolation, hygiene, and personal protection allowed containment of the SARS outbreak in Hanoi.

[Influenza: interspecies transmissions and viral rearrangement]. / Grippe: transmissions inter-espèces et réassortiment viral.

Influenza is an infection of humans beings and many animal species. It is caused by viruses which belong to the Orthomyxoviridae family. There are three types of influenza viruses A, B and C. The type A is the most pathogenic of all. The type is determined mainly by the nature of the nucleoprotein (NP), an antigen which does not greatly vary. On the contrary, the surface antigens, among which the haemagglutinin is the most important, are highly variable and their nature determines the sub-type of virus within the type A. The expressed mutations affecting the haemagglutinin are referred as antigenic drift and make virological surveillance necessary in order to annually assess the composition of the vaccine strains. The segmented nature of the genome of influenza viruses, makes possible the genetic reassortment of two different influenza viruses co-infecting one cell and produces a new hybrid virus. When such an event affects the haemagglutinin, the reassortment leads to an antigenic shift. In nature, it most certainly takes place in swine, between human and avian viruses. Whereas antigenic drift is a continuous and progressive phenomenon, antigenic shift occurs occasionally every 10 to 30 years. The emergence of a hybrid virus bearing a new haemagglutinin and thus belonging to a new human subtype, can be the starting point of the genesis of a pandemic, generally associated with a high mortality rate in humans. The participation of the pig is specially mentioned.

Natural infection of dogs by influenza C virus.

To date, only one seroepidemiological survey, carried out in Japan, gave a strong indication that dogs may be naturally infected by the influenza C virus, long considered to be exclusively human. In the present work, 134 serum samples were collected during the winter of 1988/89 from dogs aged 6 months to 16 years in northern France. Samples were tested for the presence of antibodies to influenza C virus by both haemagglutination inhibition (HI) and ELISA. Using antibody absorption by staphylococcal protein A, we demonstrated the specificity of the results. In 62% of cases, the results were identical using the two methods. Significant HI activity was found in 32% of the 134 tested sera and titres ranged from 20 to 320. Of the sera tested, 42% were positive by ELISA and titres ranged from 500 to 8,000. The discordant results are discussed. The population tested was divided into five age groups: less than 4 years, 4 to 6 years, 7 to 9 years, 10 to 11 years and greater than 12 years. The distribution of antibodies in the tested canine population, in contrast to that of humans, did not show a significant degree of association with age.

Analytical detection of 9(4)-O-acetylated sialoglycoproteins and gangliosides using influenza C virus.

The unique glycoprotein of influenza C virus, designated hemagglutinin (HEF), exhibits three functions: hemagglutination, esterase activity, and fusion factor. As the virus uses 9-O-acetylated sialic acid as a high-affinity receptor determinant for attachment to cells, its binding activity was used to reveal O-acetylated sialic acid residues after polyacrylamide gel electrophoresis and transfer onto nitrocellulose sheets of proteins and thin-layer chromatography of lipids. The specificity of the binding for O-acetylated sialoglycoconjugates was investigated. Our results showed that influenza C virus could detect the different forms of the two murine glycophorins which are known to be O-acetylated sialoglycoconjugates. The virus also bound to O-acetylated gangliosides isolated from embryonic chicken brain such as purified O-acetylated NeuAc alpha (2-8)NeuAc alpha (2-8)NeuAc alpha (2-3)Gal beta (1-4)Glc beta (1-1)ceramide (GT3). The esterase activity of the HEF protein of influenza C virus was used to unmask the sialic acid. After its deacetylation by the virus enzyme, the O-acetylated GT3 was recognized by a monoclonal antibody which binds only to the nonacetylated derivative. The results presented here show that influenza C virus is a discriminating analytical probe for identifying O-acetylated sialoglycoconjugates directly after Western blotting of proteins and thin-layer chromatography of lipids, thus providing a new analytical tool.

Monoclonal antibody A2B5, which detects cell surface antigens, binds to ganglioside GT3 (II3 (NeuAc)3LacCer) and to its 9-O-acetylated derivative.

The monoclonal antibody A2B5 recognizes antigens at the surface of neuronal and glial cells but also at the surface of thymus epithelia and pancreatic islet cells. Although these antigens have been characterized as polysialogangliosides, A2B5 also reacts with other unidentified gangliosides. In order to characterize further the epitope of A2B5, two new ganglioside antigens isolated from chicken brain are identified in this study. One is the ganglioside NeuAc alpha 2-8NeuAc alpha 2-8NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1ceramide (GT3) and the other is a 9-O-acetylated derivative of GT3). This derivative was purified from 10-day embryonic chicken brain. Acetyl groups substituted on sialic acid were removed either by alkali treatment or by incubation with influenza virus C, which contains receptor-destroying enzyme (a neuraminidate 9-O-acetyl esterase). The product of alkali treatment or viral action was detected by the antibody 18B8 which is specific for GT3. The deacetylated product still reacts with A2B5. These data and the results of mild oxidation of the antigen with sodium periodate suggest that the epitope recognized by antibody A2B5 contains the trisialyl structure found in GT3 but does not include the polyalcohol chain of the terminal sialic acid which can be oxidized by periodate or acetylated without modifying the affinity for the antibody. The epitope recognized by A2B5 is different from the epitope recognized by the antibody 18B8 in that 18B8 requires the three sialic acids with an intact and unsubstituted polyalcohol chain. Antibody 18B8 does not bind to 9-O-acetylated GT3 or GT3 oxidized by sodium periodate.

Activity of influenza C virus O-acetylesterase with O-acetyl-containing compounds.

Influenza C virus (strain C/Johannesburg/1/66) was grown, harvested, purified and used as source for the enzyme O-acetylesterase (N-acyl-O-acetylneuraminate O-acetylhydrolase; EC 3.1.1.53). This activity was studied and characterized with regard to some new substrates. The pH optimum of the enzyme is around 7.6, its stability at different pH values shows a result similar to that of the pH optimum, and its activity is well maintained in the pH range from 7.0 to 8.5 (all these tests were performed with 4-nitrophenyl acetate as substrate). Remarkable differences were found in the values of both Km and Vmax, with the synthetic substrates 4-nitrophenyl acetate, 2-nitrophenyl acetate, 4-methylumbelliferyl acetate, 1-naphthyl acetate and fluorescein diacetate. The use of 4-nitrophenyl acetate, 4-methylumbelliferyl acetate or 1-naphthyl acetate as substrate seems to be convenient for routine work, but it is better to carry out the measurements in parallel with those on bovine submandibular gland mucin (the latter is a natural and commercially available substrate). It was found that 4-acetoxybenzoic acid, as well as the methyl ester of 2-acetoxybenzoic acid, but not 2-acetoxybenzoic acid itself, are cleaved by this enzyme. Triacetin, di-O-acetyladenosine, tri-O-acetyladenosine, and di-O-acetyl-N-acetyladenosine phosphate, hitherto unreported as substrates for this viral esterase, are hydrolysed at different rates by this enzyme. We conclude that the O-acetylesterase from influenza C virus has a broad specificity towards both synthetic and natural non-sialic acid-containing substrates. Zn2+, Mn2+ and Pb2+ (as their chloride salts), N-acetylneuraminic acid, 4-methyl-umbelliferone and 2-acetoxybenzoic acid (acetylsalicylic acid) did not act as inhibitors.