Evaluation of formulations to improve SARS-CoV-2 viability and thermostability after lyophilisation.

In the context of the COVID-19 pandemic, virus collections such as EVA-GLOBAL play a key role in the supply of viruses and related products for research. Freeze-drying techniques for viruses represent a method of choice for the preservation of strains and their distribution without the need for a demanding cold chain. Here, we describe an optimised lyophilisation protocol usable for SARS-CoV-2 strains that improves preservation and thermostability. We show that sucrose used as an adjuvant represents a simple and efficient stabilizer providing increased protection for long-term preservation and shipment of the virus under different climatic conditions.

Influenza-induced acute respiratory distress syndrome during the 2010-2016 seasons: bacterial co-infections and outcomes by virus type and subtype.

OBJECTIVES: We aimed to describe bacterial co-infections and acute respiratory distress (ARDS) outcomes according to influenza type and subtype. METHODS: A retrospective observational study was conducted from 2012 to 2016 in patients admitted to the respiratory intensive care unit (ICU) of Marseille university hospital for influenza-induced ARDS. Microbiological investigations, including multiplex molecular respiratory panel testing and conventional bacteriological cultures, were performed as part of the routine ICU care on the bronchoalveloar lavage collected at admission. Bacterial co-infections, ICU mortality and respiratory function were investigated according to virus type and subtype. RESULTS: Among the 45 ARDS patients included, A(H1N1)pdm09 was the most frequent influenza virus identified (28/45 A(H1N1)pdm09, eight out of 45 A(H3N2) and nine out of 45 influenza B). Bacterial co-infections involving a total of 23 bacteria were diagnosed in 16/45 patients (36%). A(H1N1)pdm09 patients presented fewer bacterial co-infections (17.9% vs. 50.0% for A(H3N2) patients and 77.8% for B patients; p < 0.01). Overall, mortality at 90 days post admission was 33.3% (15/45), and there was no significant difference between influenza type and subtype. The need for extracorporeal membrane oxygenation was more frequent for A(H1N1)pdm2009 (20/28, 71.4%) and B patients (7/9, 77.8%) than the A(H3N2) subtype (1/8, 12.5%; p < 0.01). A(H1N1)pdm09-ARDS patients were associated with fewer ventilation-free days at day 28 (median (IQR): 0 (0-8) days) compared with other influenza-ARDS patients (15 (0-25) days, p < 0.05). DISCUSSION: In a population of influenza-induced ARDS, A(H1N1)pdm09 was associated with fewer bacterial co-infections but poorer respiratory outcomes. These data underline the major role of A(H1N1)pdm09 subtype on influenza disease severity.

The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade.

In 2019, a new coronavirus (2019-nCoV) infecting Humans has emerged in Wuhan, China. Its genome has been sequenced and the genomic information promptly released. Despite a high similarity with the genome sequence of SARS-CoV and SARS-like CoVs, we identified a peculiar furin-like cleavage site in the Spike protein of the 2019-nCoV, lacking in the other SARS-like CoVs. In this article, we discuss the possible functional consequences of this cleavage site in the viral cycle, pathogenicity and its potential implication in the development of antivirals.

GloPID-R report on chikungunya, o'nyong-nyong and Mayaro virus, part 5: Entomological aspects.

The GloPID-R (Global Research Collaboration for Infectious Disease Preparedness) chikungunya (CHIKV), o'nyong-nyong (ONNV) and Mayaro virus (MAYV) Working Group has been established to investigate natural history, epidemiology and clinical aspects of infection by these viruses. Here, we present a report dedicated to entomological aspects of CHIKV, ONNV and MAYV. Recent global expansion of chikungunya virus has been possible because CHIKV established a transmission cycle in urban settings using anthropophilic vectors such as Aedes albopictus and Aedes aegypti. MAYV and ONNV have a more limited geographic distribution, being confined to Africa (ONNV) and central-southern America (MAYV). ONNV is probably maintained through an enzootic cycle that has not been characterized yet, with Anopheles species as main vectors and humans as amplification hosts during epidemics. MAYV is transmitted by Haemagogus species in an enzootic cycle using non-human primates as the main amplification and maintenance hosts, and humans becoming sporadically infected when venturing in or nearby forest habitats. Here, we focused on the transmission cycle and natural vectors that sustain circulation of these viruses in their respective locations. The knowledge of the natural ecology of transmission and the capacity of different vectors to transmit these viruses is crucial to understand CHIKV emergence, and to assess the risk that MAYV and ONNV will expand on wide scale using anthropophilic mosquito species not normally considered primary vectors. Finally, the experts identified knowledge gaps and provided adapted recommendations, in order to address future entomological investigations in the right direction.

GloPID-R report on chikungunya, o'nyong-nyong and Mayaro virus, part 2: Epidemiological distribution of o'nyong-nyong virus.

The GloPID-R (Global Research Collaboration for Infectious Disease Preparedness) chikungunya (CHIKV), o'nyong-nyong (ONNV) and Mayaro virus (MAYV) Working Group has been established to identify gaps of knowledge about the natural history, epidemiology and medical management of infection by these viruses, and to provide adapted recommendations for future investigations. Here, we present a report dedicated to ONNV epidemiological distribution. Two large-scale ONNV outbreaks have been identified in Africa in the last 60 years, interspersed with sporadic serosurveys and case reports of returning travelers. The assessment of the real scale of ONNV circulation in Africa remains a difficult task and surveillance studies are necessary to fill this gap. The identification of ONNV etiology is made complicated by the absence of multiplex tools in co-circulation areas and that of reference standards, as well as the high cross-reactivity with related pathogens observed in serological tests, in particular with CHIKV. This is a specific obstacle for seroprevalence studies, that necessitate an improvement of serological tools to provide robust results. The scarcity of existent genetic data currently limits molecular epidemiology studies. ONNV epidemiology would also benefit from reinforced entomological and environmental surveillance. Finally, the natural history of the disease deserves to be further investigated, with a specific attention paid to long-term complications. Considering our incomplete knowledge on ONNV distribution, GloPID-R CHIKV, ONNV and MAYV experts recommend that a major effort should be done to fill existing gaps.

Risk factors of exposure to Aedes albopictus bites in mainland France using an immunological biomarker.

In recent decades, the invasive Aedes albopictus vector has spread across Europe and is responsible for numerous outbreaks of autochthonous arboviral disease. The aim of this study was to identify epidemiological and sociological risk factors related to individual levels of exposure to Aedes albopictus bites. A multidisciplinary survey was conducted with volunteer blood donors living in areas either colonised or not by Aedes albopictus in mainland France. Individual levels of exposure were evaluated by measuring the IgG level specific to Aedes albopictus saliva. The most striking risk factors concerned the localisation and characteristics of the dwelling. Individuals living in areas colonised prior to 2009 or recently colonised (between 2010 and 2012) had higher anti-salivary gland extract IgG levels compared with those who were living in areas not yet colonised by Ae. albopictus. The type of dwelling did not seem to impact the level of exposure to Aedes bites. People living in apartments had a higher anti-salivary gland extract IgG level than those living in individual houses but the difference was not statistically significant. Interestingly, the presence of air conditioning or window nets was associated with a noticeable reduction in bite intensity.

GloPID-R report on Chikungunya, O'nyong-nyong and Mayaro virus, part I: Biological diagnostics.

The GloPID-R (Global Research Collaboration for Infectious Disease Preparedness) Chikungunya (CHIKV), O'nyong-nyong (ONNV) and Mayaro virus (MAYV) Working Group is investigating the natural history, epidemiology and medical management of infection by these viruses, to identify knowledge gaps and to propose recommendations for direct future investigations and rectification measures. Here, we present the first report dedicated to diagnostic aspects of CHIKV, ONNV and MAYV. Regarding diagnosis of the disease at the acute phase, molecular assays previously described for the three viruses require further evaluation, standardized protocols and the availability of international standards representing the genetic diversity of the viruses. Detection of specific IgM would benefit from further investigations to clarify the extent of cross-reactivity among the three viruses, the sensitivity of the assays, and the possible interfering role of cryoglobulinaemia. Implementation of reference panels and external quality assessments for both molecular and serological assays is necessary. Regarding sero-epidemiological studies, there is no reported high-throughput assay that can distinguish among these different viruses in areas of potential co-circulation. New specific tools and/or improved standardized protocols are needed to enable large-scale epidemiological studies of public health relevance to be performed. Considering the high risk of future CHIKV, MAYV and ONNV outbreaks, the Working Group recommends that a major investigation should be initiated to fill the existing diagnostic gaps.

Exploratory re-encoding of yellow fever virus genome: new insights for the design of live-attenuated viruses.

Virus attenuation by genome re-encoding is a pioneering approach for generating effective live-attenuated vaccine candidates. Its core principle is to introduce a large number of synonymous substitutions into the viral genome to produce stable attenuation of the targeted virus. Introduction of large numbers of mutations has also been shown to maintain stability of the attenuated phenotype by lowering the risk of reversion and recombination of re-encoded genomes. Identifying mutations with low fitness cost is pivotal as this increases the number that can be introduced and generates more stable and attenuated viruses. Here, we sought to identify mutations with low deleterious impact on the in vivo replication and virulence of yellow fever virus (YFV). Following comparative bioinformatic analyses of flaviviral genomes, we categorised synonymous transition mutations according to their impact on CpG/UpA composition and secondary RNA structures. We then designed seventeen re-encoded viruses with 100-400 synonymous mutations in the NS2A-to-NS4B coding region of YFV Asibi and Ap7M (hamster-adapted) genomes. Each virus contained a panel of synonymous mutations designed according to the above categorisation criteria. The replication and fitness characteristics of parent and re-encoded viruses were compared in vitro using cell culture competition experiments. In vivo laboratory hamster models were also used to compare relative virulence and immunogenicity characteristics. Most of the re-encoded strains showed no decrease in replicative fitness in vitro. However, they showed reduced virulence and, in some instances, decreased replicative fitness in vivo. Importantly, the most attenuated of the re-encoded strains induced robust, protective immunity in hamsters following challenge with Ap7M, a virulent virus. Overall, the introduction of transitions with no or a marginal increase in the number of CpG/UpA dinucleotides had the mildest impact on YFV replication and virulence in vivo. Thus, this strategy can be incorporated in procedures for the finely tuned creation of substantially re-encoded viral genomes.

The European Virus Archive goes global: A growing resource for research.

The European Virus Archive (EVA) was created in 2008 with funding from the FP7-EU Infrastructure Programme, in response to the need for a coordinated and readily accessible collection of viruses that could be made available to academia, public health organisations and industry. Within three years, it developed from a consortium of nine European laboratories to encompass associated partners in Africa, Russia, China, Turkey, Germany and Italy. In 2014, the H2020 Research and Innovation Framework Programme (INFRAS projects) provided support for the transformation of the EVA from a European to a global organization (EVAg). The EVAg now operates as a non-profit consortium, with 26 partners and 20 associated partners from 21 EU and non-EU countries. In this paper, we outline the structure, management and goals of the EVAg, to bring to the attention of researchers the wealth of products it can provide and to illustrate how end-users can gain access to these resources. Organisations or individuals who would like to be considered as contributors are invited to contact the EVAg coordinator, Jean-Louis Romette, at jean-louis.romette@univmed.fr.

Cytomegalovirus reactivation enhances the virulence of Staphylococcus aureus pneumonia in a mouse model.

OBJECTIVES: Cytomegalovirus (CMV) reactivation in intensive care unit patients may increase mortality and favour bacterial pneumonia. We developed a murine model to compare the severity of staphylococcal pneumonia after CMV reactivation and in CMV-negative mice. METHODS: Balb/c mice were primo-infected with murine cytomegalovirus (MCMV n=90) or received saline (control n=90). After latency, all mice underwent caecal ligation and puncture to trigger MCMV reactivation in MCMV primary-infected mice. Surviving animals received an intra-nasal inoculation with methicillin-susceptible Staphylococcus aureus (MSSA) to induce pneumonia. Mortality, lung bacterial count, histology and interferon-alpha and gamma serum levels were compared in MCMV reactivated and control mice 2, 5 and 15 days after pneumonia. RESULTS: After MSSA pneumonia, MCMV mice showed a trend towards a higher mortality (9.4% versus 0%; p 0.09) and a higher weight loss (2.2 (0.6-4.1 g) versus 0.7 (-0.3 to 1.3 g); p 0.005). The lung bacterial count was higher in MCMV mice 2 days (5×103 (103 to 3×105) versus 102 (0 to 4×102) CFU/lung; p 0.007) and 5 days (2.5×104 (1.6×104 to 6.5×105) versus 15 (10-40) CFU/lung; p 0.005) after MSSA pneumonia. 8/40 (20%) MCMV mice developed lung abscesses compared to 0% in control (p 0.011). Interferon-alpha serum levels 2 days after staphylococcal pneumonia were higher in MCMV mice. CONCLUSIONS: MCMV reactivation decreased lung bacterial clearance and favoured the development of staphylococcal abscessing pneumonia. CMV reactivation may be responsible for a higher susceptibility to bacterial sepsis.

Immunity against influenza A(H1N1) infections is determined by age at the time of initial strain circulation.

We explored age-dependent patterns in haemagglutination inhibition (HI) titre to seasonal [1956 A(H1N1), 1977 A(H1N1), 2007 A(H1N1)] and pandemic [A(H1N1)pdm09] influenza strains using serological data collected from an adult French influenza cohort. Subjects were recruited by their general practitioners from 2008 to 2009 and followed until 2010. We explored age-related differences between strain-specific HI titres using 1053 serological samples collected over the study period from 398 unvaccinated subjects. HI titres against the tested seasonal and pandemic strains were determined using the HI technique. Geometric mean titres (GMTs) were estimated using regression models for interval-censored data. Generalized additive mixed models were fit to log-transformed HI estimates to study the relationship between HI titre and age (age at inclusion and/or age at initial strain circulation). GMT against one strain was consistently highest in the birth cohort exposed to that strain during childhood, with peak titres observed in subjects aged 7-8 years at the time of initial strain circulation. Our results complete previous findings on influenza A(H3N2) strains and identify a strain-dependent relationship between HI titre and age at initial strain circulation.

Complete coding sequence of Zika virus from Martinique outbreak in 2015.

Zika virus is an Aedes-borne Flavivirus causing fever, arthralgia, myalgia rash, associated with Guillain-Barré syndrome and suspected to induce microcephaly in the fetus. We report here the complete coding sequence of the first characterized Caribbean Zika virus strain, isolated from a patient from Martinique in December, 2015.

Virus isolation, genetic characterization and seroprevalence of Toscana virus in Algeria.

Toscana virus (TOSV; Bunyaviridae, Phlebovirus) is transmitted by sandflies of the genus Phlebotomus in the Mediterranean area. One strain of TOSV was isolated from a total of almost 23 000 sandflies collected in Kabylia, Algeria. The complete genome was sequenced, and phylogenetic studies indicated that it was most closely related with TOSV strain from Tunisia within lineage A, which also includes Italian, French and Turkish strains. A seroprevalence study performed on 370 sera collected from people living in the same area showed that almost 50% possessed neutralizing antibodies against TOSV, a rate much higher than that observed in Southern Europe. Sandfly species distribution in the study area suggests that the vector of TOSV in this region belongs to the subgenus Larroussius. These data support the rapid implementation of the diagnosis of TOSV in clinical microbiology laboratories to estimate the burden in patients presenting with neuroinvasive infections and febrile illness.

Infection of sand flies collected from different bio-geographical areas of Tunisia with phleboviruses.

An entomological investigation performed in 2013 covering different bio-geographical areas varying from humid in the north to the arid in the center showed that sand flies of the subgenus Larroussius including Phlebotomus perniciosus, Phlebotomus perfiliewi, and Phlebotomus longicuspis are abundant and widely distributed in Tunisia. A total of 3992 collected and pooled with up to 30 specimens per pool based on sex, trapping location and collection data were tested for the presence of phleboviruses by nested reverse transcriptase polymerase chain reaction and sequencing. Of a total of 135 pools, 23 were positive, yielding and minimum infection rate of 0.6%. Phylogenetic analysis performed using partial amino acid sequence in the polymerase gene showed that all these phleboviruses were grouped in one cluster clearly distinct from but closely related to Massilia virus and Granada virus. This putative novel virus, tentatively called Saddaguia virus (SADV), is widely distributed in Tunisia. Together with Toscana, Punique, and Utique viruses, SADV is the fourth recognized phlebovirus to be transmitted by sand flies in Tunisia. The medical and public health interest of SADV remains to be investigated.

Arthropods as a source of new RNA viruses.

The discovery and development of methods for isolation, characterisation and taxonomy of viruses represents an important milestone in the study, treatment and control of virus diseases during the 20th century. Indeed, by the late-1950s, it was becoming common belief that most human and veterinary pathogenic viruses had been discovered. However, at that time, knowledge of the impact of improved commercial transportation, urbanisation and deforestation, on disease emergence, was in its infancy. From the late 1960s onwards viruses, such as hepatitis virus (A, B and C) hantavirus, HIV, Marburg virus, Ebola virus and many others began to emerge and it became apparent that the world was changing, at least in terms of virus epidemiology, largely due to the influence of anthropological activities. Subsequently, with the improvement of molecular biotechnologies, for amplification of viral RNA, genome sequencing and proteomic analysis the arsenal of available tools for virus discovery and genetic characterization opened up new and exciting possibilities for virological discovery. Many recently identified but "unclassified" viruses are now being allocated to existing genera or families based on whole genome sequencing, bioinformatic and phylogenetic analysis. New species, genera and families are also being created following the guidelines of the International Committee for the Taxonomy of Viruses. Many of these newly discovered viruses are vectored by arthropods (arboviruses) and possess an RNA genome. This brief review will focus largely on the discovery of new arthropod-borne viruses.

Clinical significance of intra-host variability of Dengue-1 virus in venous and capillary blood.

Dengue fever represents a major public health problem. Both viral and host immune factors are involved in severe infections. Humans and mosquito-vectors are infected with diverse viral populations that may play a role in viral adaptation and disease pathogenesis. Our objective was to analyse the intra-host genetic variability of dengue virus type 1 (DENV-1) in the venous and capillary blood and its relationships with the clinical presentation of dengue fever. Early serum samples were collected in 2009 from ten DENV-1-infected patients hospitalized in Santa Cruz de la Sierra, Bolivia. Partial viral envelope sequences were analysed at the inter-host and intra-host level. For each patient, an average of 56 clone sequences was analysed both in the venous sector and the capillary sector (from right and left hands). The ten consensus sequences were highly similar. The intra-host DENV-1 genetic variability was significantly lower in the venous sector than in the capillary sector, and in patients with haemorrhagic symptoms than in those without haemorrhagic symptoms, particularly in capillary samples. No relation was found with sex, age, dengue IgG-serological status, day of serum sampling, or viral load. Significant relationships were found between the clinical presentation of dengue fever and the variability of viral populations within hosts, particularly in capillary samples. The observed variability of envelope sequences at the early phase of dengue infection was not critically influenced by the previous dengue serological status of patients. An important part of viral microevolution may occur in the capillary sector and influence the mechanisms of severe forms.