Phylogeography of Coxsackievirus A16 Reveals Global Transmission Pathways and Recent Emergence and Spread of a Recombinant Genogroup.

Coxsackievirus A16 (CV-A16; Picornaviridae) is an enterovirus (EV) type associated with hand, foot, and mouth disease (HFMD) in children. To investigate the spatial spread of CV-A16, we used viral sequence data sampled during a prospective sentinel surveillance of HFMD in France (2010 to 2014) and phylogenetic reconstruction. A data set of 168 VP1 sequences was assembled with 416 publicly available sequences of various geographic origins. The CV-A16 sequences reported were assigned to two clades, genogroup B and a previously uncharacterized clade D. The time origins of clades B and D were assessed in 1978 (1973 to 1981) and 2004 (2001 to 2007), respectively. The shape of the global CV-A16 phylogeny indicated worldwide cocirculation of genetically distinct virus lineages over time and across geographic regions. Phylogenetic tree topologies and Bayes factor analysis indicated virus migration. Virus transportation events in clade B within Europe and Asia and between countries of the two geographic regions were assessed. The sustained transmission of clade D viruses over 4 years was analyzed at the township level in France and traced back to Peru in South America. Comparative genomics provided evidence of recombination between CV-A16 clades B and D and suggested an intertype recombinant origin for clade D. Time-resolved phylogenies and HFMD surveillance data indicated that CV-A16 persistence is sustained by continuing virus migration at different geographic scales, from community transmission to virus transportation between distant countries. The results showed a significant impact of virus movements on the epidemiological dynamics of HFMD that could have implications for disease prevention.IMPORTANCE Coxsackievirus A16 is one of the most prevalent enterovirus types in hand, foot, and mouth disease outbreaks reported in Southeast Asia. This study is based on epidemiological and viral data on HFMD caused by CV-A16 in a European country. The phylogeographic data complemented the syndromic surveillance with virus migration patterns between geographic regions in France. The results show how viral evolutionary dynamics and global virus spread interact to shape the worldwide pattern of an EV disease. CV-A16 transmission is driven by movements of infected individuals at different geographic levels: within a country (local dynamics), between neighboring countries (regional dynamics), and between distant countries (transcontinental dynamics). The results are consistent with our earlier data on EV-A71 and confirm the epidemiological interconnection of Asia and Europe with regard to EV infections.

First record of the Asian bush mosquito, Aedes japonicus japonicus, in Italy: invasion from an established Austrian population.

BACKGROUND: In 2011 we identified the Asian bush mosquito, Aedes japonicus japonicus (Theobald, 1901) (Diptera: Culicidae) for the first time in northern Slovenia and in the bordering Austrian federal state of Styria. Between May and July 2012 the distribution area of Ae. j. japonicus was already found to be extended westwards into Carinthia and eastwards towards Burgenland and bordering Hungary. In August 2012 the species was first detected in a western province of Hungary. In subsequent years, follow-up field studies demonstrated an active spread westwards throughout Carinthia, reaching the border to northern Italy. FINDINGS: In July 2015 several aquatic-stage specimens of the species were discovered at three different sites in the Friuli Venezia Giulia region, north-eastern Italy. In September 2015, co-occurrence of Ae. j. japonicus and Aedes albopictus (Skuse, 1895) was observed in the same sample in that region. CONCLUSIONS: Ae. j. japonicus actively extended its geographic range from an established population in Carinthia (Austria) southwards to northern Italy by crossing Alpine ranges. Since Ae. albopictus and Aedes koreicus (Edwards, 1917) are already well established in northern Italy, it will be pivotal to monitor the consequences of a third invasive mosquito species trying to populate the same geographic region.

Transmission patterns of human enterovirus 71 to, from and among European countries, 2003 to 2013.

Enterovirus 71 (EV-71) is involved in epidemics of hand, foot, and mouth disease (HFMD) and has been reported to occur with severe neurological complications in eastern and south-east Asia. In other geographical areas, the transmission of this virus is poorly understood. We used large sequence datasets (of the gene encoding the viral protein 1, VP1) and a Bayesian phylogenetic approach to compare the molecular epidemiology and geographical spread patterns of EV-71 subgenogroups B4, B5, C1, C2, and C4 in Europe relative to other parts of the world. For the study, European countries considered were European Union (EU) Member States and Iceland, Norway and Switzerland. Viruses of the B4, B5, and C4 subgenogroups circulate mainly in eastern and south-east Asia. In Europe sporadic introductions of these subgenogroups are observed, however C1 and C2 viruses predominate. The phylogenies showed evidence of multiple events of spread involving C1 and C2 viruses within Europe since the mid-1990s. Two waves of sporadic C2 infections also occurred in 2010 and 2013. The 2007 Dutch outbreak caused by C2 and the occurrence of B5 and C4 infections in the EU between 2004 and 2013 arose while the circulation of C1 viruses was low. A transmission chain involving a C4 virus was traced from Japan to the EU and then further to Canada between 2001 and 2006. Recent events whereby spread of viruses have occurred from, to, and within Europe appear to be involved in the long term survival of EV-71, highlighting the need for enhanced surveillance of this virus.

Antagonistic antiviral activity between IFN-lambda and IFN-alpha against lethal Crimean-Congo hemorrhagic fever virus in vitro.

BACKGROUND AND AIMS: Crimean Congo Hemorrhagic fever virus (CCHFV) is the causative agent of Crimean-Congo hemorrhagic fever, a severe disease with a mortality rate of around 30% in humans. Previous studies demonstrate that pre-treatment with type I IFNs have an antiviral effect against CCHFV, while established CCHFV infection is almost insensitive to subsequent IFN-α treatment. No data concerning type III IFNs antiviral activity against CCHFV are available so far. The aim of the present study was to explore the capability of IFN-λ1 to inhibit the replication of CCHFV and the possible synergism/antagonism between IFN-α and IFN-λ1 both in the inhibition of CCHFV replication and in the activation of intracellular pathways of IFN response. METHODS: Human A549 and HuH7 cells were treated with increasing amounts of IFN-λ1, or IFN-α or a combination of them, infected with CCHF; the extent of virus yield inhibition and the induction of MxA and 2'-5'OAS mRNA was measured. RESULTS AND CONCLUSIONS: Our study pointed out that type III IFN possess an antiviral activity against CCHFV, even if lower than type I IFN. Moreover, a clear antagonism between IFN-λ and IFN-α was observed in both cell lines (A549 and HuH7 cells), in terms of antiviral effect and activation of pivotal ISGs, i.e. MxA and 2'-5'OAS. Elucidating the interplay between type I and III IFNs will help to better understand innate defence mechanisms against viral infections and may provide novel scientific evidence for a more rational planning of available and future treatments, particularly against human diseases caused by high concern viruses.

Serological evidence for Parapoxvirus infection in chamois from the Tyrol regions of Austria and Italy.

Orf-virus (ORFV) is a parapoxvirus that infects small ruminants worldwide causing sporadic zoonotic infections, mainly transmitted by direct contact with sheep and goats. Following an ORFV case in a hunter of Alpine chamois (Rupicapra rupicapra), who did not report previous contact to domestic animals, a serological survey in Western Austria was conducted to assess the seroprevalence of ORFV in this species. In addition, this study also tested blood/tissue samples of chamois from different areas of the adjacent province of Bolzano/Northern Italy for antibodies against ORFV using immunofluorescence and ELISA. The observed seropositivity rates in the chamois tested on the Austrian and Italian side of the Alps were 23.5% and 9.5%, respectively, with a combined 95% confidence interval ranging from 0.0678 to 0.238. Although the prevalence was significantly lower than the one observed in Austrian sheep flocks, this study provided the first evidence that parapoxviruses have spilled over into chamois populations to a significant degree in the Tyrol regions of Austria and Italy.

Protein kinase C overexpression does not enhance immune-stimulatory surface markers of vaccinia-infected dendritic cells and DC cell lines.

One of the shortcomings of vaccinia virus (VACV) as immunization vector is the down-regulation of HLA and costimulatory molecules in antigen presenting cells. To overcome this problem we investigated the use of protein kinase C (PKC) as immune stimulatory agent. Thus several classical and atypical PKCs were inserted into wild-type or attenuated VACV using recombination into the hemagglutinin gene and the expression driven by the VACV 7,5K-IE gene promoter. Recombinant constructs expressing PKC-alpha, -beta, -theta as well as wild-type, constitutive active or dominant negative PKC-zeta constructs were generated. Additional constructs expressing PKB/Akt1 and ICAM-1 were used for comparison. Immature and mature peripheral blood derived-dendritic cells (DC) as well as lymphoid cell lines capable of obtaining a DC-like phenotype upon mitogen stimulation were infected. Disappointingly, VACV-driven PKC overexpression did not significantly enhance expression of various activation markers or costimulatory molecules tested. Neither CD86 nor HLA-DR expression was upregulated and also no influence on the maturation of DC, as measured by DC-SIGN and CD83, was observed. However, VACV did not interfere with LPS induced up-regulation of CD83 and did not lead to substantial apoptosis of infected DC within the first 24 hours.

Evaluation of a "home-made" method to determine viral genotype and characterize mutations conferring drug resistance in chronic hepatitis B patients.

We compared a home-made sequencing system to analyze plasma samples from patients with chronic HBV infection with the commercial TRUGENE(®) HBV Genotyping Assay. A PCR and sequencing protocol based on published primers was applied to detect the viral genotypes as well as the major patterns of point mutations leading to resistance to lamivudine, adefovir and entecavir. For the determination of HBV genotypes the obtained sequences were aligned with a database created within the RIDOM TraceEdit program and publicly available reference sequences. Our results showed perfect correlation with the commercial system, with types D (72%) and A (22%) being the most frequent genotypes. The resistance loci were also reliably detected with mostly combined L180M and M204V/I mutations as the local patterns. M204I mutations were more frequent in genotype D, M204V in genotype A isolates. G173L mutations were not found. The only genotype C isolate tested revealed a different pattern (E263D and I269L). These data speak for the usability of this rapid amplification and sequencing approach for routine genotyping of HBV isolates and simultaneous determination of the drug resistance profile of the dominant viral species.

Cowpox virus isolate virulent in humans shows attenuated phenotype in mice.

We have cultured Cowpox virus (CPXV) from skin lesion material of a human patient from Austria. Phylogenetic comparison of the HA-gene revealed a rather homogeneous cluster with other local isolates from recent years, the A36R-gene was mostly related to elephant derived strains from Germany. Despite causing disease in human, the isolate AT/Carinthia/788/07 surprisingly even at high titers showed a highly reduced virulence in BALB/c mice upon intranasal inoculation as compared to vaccinia virus. This contrasts earlier reports on other CPXV isolates. Using shotgun DNA sequencing several insertions and deletions were found in genes presumably involved in host range, immune regulation as well as established virulence factors. These preliminary data could be an indication that CPXV strains with proven pathogenicity for humans may have reduced virulence in mice and vice versa. Additionally strains with a reduced virulence may have an advantage in persisting in less dense rodent populations.

Investigation of bacterial and viral agents and immune status in Behcet's disease patients from Iran.

AIM: Behçet's disease (BD) is an autoimmune disorder associated with HLA-B51 positivity. Serologic/genomic findings have suggested microbes as possible causative agents and the geographical distribution suggests environmental influences. METHODS: We performed comparative analyses of 40 patients with BD or related symptoms not fulfilling BD criteria. Patients originating from different regions of Iran were tested by molecular/serological methods for human herpes viruses and parvovirus B19, two Chlamydiae species, as well as Coxiella, Listeria, Yersinia, Leptospira and Mycobacterium paratuberculosis. Human leukocyte antigen-typing was performed: testing of cytokine profiles and immune mediators representative for the cellular immune system, including neopterin/kynurenine production. RESULTS: No apparent differences in interleukin (IL)-4, 6, 8 and 10 were observed, whereas production of soluble IL-2-receptor and tumor necrosis factor (TNF)-alpha were more pronounced in the BD group. Neopterin/kynurenine production was comparable, although both groups showed twice the levels of healthy people. No significant differences of herpes simplex virus (HSV) antibody titres were observed but higher titres against Chlamydophila pneumoniae were found in the controls. In 20 BD patients and controls neither parvovirus B19 DNA was detected nor bacterial DNA. Viral DNA of Epstein-Barr virus (EBV), cytomegalovirus (CMV) and human herpes virus (HHV)8 was detected more frequently in the BD group, whereas HSV DNA was only found in the controls, indicating that stomatitis might be caused by HSV. CONCLUSION: Although no significant association of BD was detected with a single pathogen, our findings suggest that detection of HSV DNA or Chlamydiae would rather argue against classic BD. Whether there is a discriminative potential of the tested immune mediators/receptors has to be elucidated in further studies.

Dominance of CTX-M group 1 beta-lactamase enzymes in ESBL producing E. coli from outpatient urines in neighboring regions of Austria and Italy.

The importance of extended spectrum ß-lactamases (ESBL) is increasing worldwide. ESBLs of the CTX-M type are on the rise in Europe, not only in the hospital environment but also in outpatients. Therefore we performed a comparative pilot study including ESBL producing Escherichia coli isolated from outpatients suffering from urinary tract infections, 28 from Innsbruck, Austria, and 34 from Bolzano, Italy. Using established PCR methods we detected in nearly 90% of ESBL producing E. coli isolates CTX-M group 1 enzymes and only a few group 2 or group 9 enzymes. bla (TEM), bla (OXA-1) and aminoacyltransferase aac(6')-lb were significantly more frequent in the Austrian region, where also bla (SHV )was found in one isolate. In 2009 the overall prevalence of ESBL in E. coli causing urinary tract infection in outpatient samples was 7.6% in a local laboratory in Innsbruck and 5% in Bolzano. Additionally, we investigated plasmid-mediated qnr genes which can contribute to quinolone resistance, qnrA was found in an AmpC producing E. coli from Innsbruck and qnrS in two ESBL producers from Bolzano. Data confirmed that ESBL-producing E. coli have emerged as important pathogens in urinary tract infections of outpatients in both regions.

Cannabinoids lead to enhanced virulence of the smallpox vaccine (vaccinia) virus.

Indian hemp is used since thousands of years as herbal drug. We found that a single dose of cannabis resin was equally active as Δ9-tetrahydrocannabinol (THC) enhancing severity and duration of symptoms in vaccinia virus infected mice. Cowpox virus did not cause symptomatic disease, but some reduction of specific antibody production was observed in drug treated animals. In vitro cannabis was superior to THC alone at inhibiting mitogen stimulated proliferation of human and mouse spleen cells and peripheral blood mononuclear cells. Also resin sub-fractions other than THC, cannabidiol and cannabinol, recovered also from cigarette smoke, were found inhibitory, suggesting additional involvement of constituents other than psychoactive THC. The immunoregulatory effects must be differentiated from apoptotic effects on spleen cells and lymphocytic mouse cell lines, which were observed with resin and THC but not with cannabidiol or cannabinol. A significant contribution of cytotoxic effects seems unlikely as drug treated lymphocytes were still capable of producing cytokines after T-cell receptor-specific stimulation. Considering a recent case of unusually severe cowpox virus infection in a young drug taker these data confirm a risk of "soft drugs" for acquiring poxvirus infection or enhancing side effects of the smallpox vaccine and perhaps also other live vaccines.

Obstacles of Multiplex Real-Time PCR for Bacterial 16S rDNA: Primer Specifity and DNA Decontamination of Taq Polymerase.

BACKGROUND: The detection of a broad range of bacteria by PCR is applied for the screening of blood and blood products with special attention to platelet concentrates. For practical use it is desirable that detection systems include Gram-positive, Gram-negative and non-Gram-stainable bacteria. It is quite challenging to achieve high sensitivity along with a clear negative control with PCR reagents, because especially Taq polymerase is contaminated with traces of bacterial DNA. METHODS: Bacterial DNA decontamination of Taq polymerase was attempted by two different methods using the restriction enzyme Sau 3A1 and microfiltration. Additionally a commercially available Taq polymerase depleted of bacterial DNA was included. A published real-time PCR specific for Gram-negative bacteria was adapted for Gram-positive bacteria, including certain Staphylococcus species and Mycobacteria, and was used to charge the three Taq polymer-ases depleted of bacterial DNA contamination RESULTS: Despite published reports about successful DNA decontamination, all three approaches performed poorly in experiments done in this study. Sensitivity ranged at approximately 50-100 colony forming units (CFU) per PCR reaction for Escherichia coli and Staphylococcus epidermidis, corresponding to 1,250-2,500 CFU/ml sample material. Conclusion: It seems unsatisfying to accept detection limits that high for diagnostic bacterial PCR even if highly multiplexed. Reliable methods for DNA decontamination of Taq polymerase are needed and would present one important step towards bacterial DNA detection with high sensitivity.

Diazepam leads to enhanced severity of orthopoxvirus infection and immune suppression.

Benzodiazepines are drugs widely used as tranquilizers and in various other indications. We treated Balb/c mice with diazepam and infected them with cowpox (CPXV) and vaccinia virus (VACV). Disease index, weight loss and the antibody response were determined. Additionally the influence of different benzodiazepines on the mitogen response of human peripheral blood lymphocytes and spleen cells was tested. Diazepam led to earlier disease onset, prolonged duration of symptoms, higher weight loss and overall disease index in VACV infected mice. CPXV infected mice developed poxviral skin lesions only after drug administration and a significant decrease in the specific antibody response was also observed. Diazepam and alprazolam also inhibited the proliferative response of human lymphocytes/spleen cells in vitro but did not show noteworthy apoptotic effects. It is surprising that even a single dose of diazepam has a profound influence on the immune system, sufficient to facilitate symptomatic infectious disease. These data provide first evidence that commonly used drugs like Valium may augment severity of rare poxvirus infections such as CPXV or monkeypox. As VACV is still used as life vaccine against smallpox there is also a risk of enhanced side effects or possible interference with the success of vaccination.

Active in vitro reduction of antigen presenting cells in human corneal grafts using different chemokines.

BACKGROUND: For the fate of a graft the antigen presenting cells play an important role. Chemokines can lead to enhanced migration of these cells. We therefore investigated if fresh human corneas bear the chemokine receptor 7 (CCR7) and if its ligands can force the emigration of dendritic cells in an in vitro model. METHODS: We used human corneas excluded for transplantation and performed migration tests using chemokine ligands 19 (CCL19) and 21 (CCL21) or the complement factor 5a (C5a). Emigrated cells were collected up to 35 days and stained by immunofluorescent double labeling in triple layer technique with Langerin/CD207, DC-SIGN/CD209, CD14, and HLA-DR. In parallel, fresh and cultured human corneas were stained for CCR7. RESULTS: We found in fresh human corneas, as well as in long-term cultured ones, a low CCR7 expression that nearly diminished after 28 days. In vitro Langerhans cell emigration could be enhanced only by CCL19, whereas dendritic cells were strongly influenced by CCL19, CCL21, and C5a. HLA-DR(+) cells showed numerically the highest in vitro emigration rate. Macrophages/monocytes were not influenced by the used chemokines. CONCLUSIONS: Although human corneas reduce their antigen presenting cells numbers during long-term culture, this effect could be significantly enhanced by using chemokines.

In vitro prevention of vaccinia and herpesvirus infection spread in explanted human corneas by N-chlorotaurine.

OBJECTIVE: Weak oxidants produced by activated human leukocytes are proven antimicrobial substances. We tested whether N-chlorotaurine (NCT, taurine chloramine), the chlorinated metabolite of the amino acid taurine, in addition to direct virucidal effects on viral suspensions, has the capability to prevent cell-to-cell spread of viruses in human corneal epithelium. METHODS: Human corneal grafts were infected in vitro with poxvirus (vaccinia virus, VV) and herpesvirus. Different NCT dilutions were added to prevent viral spread within the corneal epithelium as detected by immune-staining and microscopy of cytopathic effects. Additionally, virus release was measured by cell culture. RESULTS: Addition of NCT significantly reduced the number of VV-infected epithelial cells at concentrations as low as 0.01% in culture medium, which was far beyond cytotoxic concentrations in long-term cultures. The release of virus by the infected corneal grafts was reduced by 2-3 log(10 )as well. As expected, herpesvirus infection was also positively affected. CONCLUSION: Smallpox has been known as a major cause of blindness in historical outbreaks. NCT could therefore provide an additional supportive means for treating orthopoxvirus-associated keratitis. Additionally, biocompatible local antiseptics like NCT could also serve as an experimental treatment in other keratitis of suspected viral origin.

Generalized parapoxvirus infection associated with increased antibody titres for varicella zoster virus and measles.

Human parapoxvirus infections are rare, self-limiting, zoonotic diseases. A 35-year-old veterinarian presented with a generalized rash of large umbilicated vesicles that appeared after antibiotic treatment for erysipelas on the forearm. The erysipelas arose from an erupted pustular thumb lesion that appeared after examining a sheep. An outbreak of chickenpox in the village suggested parapoxvirus or varicella zoster virus (VZV) was the most likely agent. No poxvirus was detected by electron microscopy or in cell cultures from lesion material. PCR revealed parapoxvirus DNA with a sequence similar to orf-viruses from Finland. Orf-virus immunofluorescence showed a titre increase, supporting the parapoxvirus diagnosis. VZV was not detected by PCR, but varicella antibodies increased three-fold in serum samples drawn two weeks apart. In addition, the patient had high antibody titres for measles and reported recent contact with individuals exposed to an outbreak of measles in nearby Austria. To explain the unusually generalized symptoms in this young and healthy patient, these findings could be variously interpreted as: i) a booster by community VZV infections; ii) a subclinical VZV (re)infection that was superinfected by the parapoxvirus; iii) an orf-virus mediated immune stimulation; iv) a post-infectious syndrome; or v) a temporary immunosuppression by subclinical measles.

Epidemiology of enterovirus types causing neurological disease in Austria 1999-2007: detection of clusters of echovirus 30 and enterovirus 71 and analysis of prevalent genotypes.

Between 1999 and 2007 1,388 stool specimens from patients with acute flaccid paralysis or aseptic meningitis were submitted to the Austrian reference laboratory for poliomyelitis. Samples (201) yielded non-poliovirus enterovirus in culture. One hundred eighty-one viruses were available for typing and 78 isolates which remained serologically untyped were further analyzed by CODEHOP-PCR and sequencing of the VP1 gene and the 5'-untranslated region (5'-UTR). Typing revealed an Echovirus 30 outbreak in northwestern Austria in 2000, which was in accordance with the situation in Europe, and no dramatic seasonal changes of Coxsackie viruses were observed. In 2002/2003 a small outbreak of enterovirus 71 (EV71), affected 12 patients in the province of Styria. This virus was identified as genotype C1 and appeared to be genetically distinct from the isolates observed in 2001/2002 in Vienna. In 2004 two unrelated cases occurred in Lower Austria, which were identified as genotype C4, which has been described associated with high mortality most recently in China. In contrast to the situation in Asia the detected EV71 cases were not associated with hand-foot-mouth disease, but with serous meningitis only. This was surprising as a recent publication suggested a reduced neurovirulence of C1 genotype in children in Norway, presumably due to alterations in 5'-UTR and polymerase gene. However, comparing the 5'-UTR of the Austrian isolates and established virulent reference strains to the Norwegian isolate and an attenuated EV71 laboratory strain we did not find an indication that the genotype C1 possesses a RNA structure in its 5'-UTR leading to reduced neurovirulence.

Involvement of protein kinase C in phagocytosis of human retinal pigment epithelial cells and induction of matrix metalloproteinase secretion.

Protein kinase C (PKC) is involved in cell activation. We investigated PKC-mediated pathways and secretion of matrix metalloproteinases (MMPs) in phagocytosis by human retinal pigment epithelial cells (RPE). We used time-resolved fluorometry for europium-labeled microsphere uptake and gel zymography to assay the influence of PKC modulators. PKC inhibitors blocked phagocytosis by RPE. ARPE-19, a human RPE-cell line, showed reduced secretion of MMP-2, although MMP-9 secretion by PKC activation was conserved in both cell types, namely in the primary RPEs and in the RPE-cell line. Particle uptake by RPE cells requires activation of PKC; the use of PKC inhibitors as new anticancer drugs may possibly cause ocular side-effects.