Inactivation mechanisms of human adenovirus by e-beam irradiation in water environments.

This study aims to study the kinetics and mechanisms of human adenovirus inactivation by electron beam. Human adenovirus type 5 (HAdV-5) was inoculated in two types of aqueous substrates (phosphate-buffered saline - PBS, domestic wastewater - WW) treated by electron beam at a dose range between 3 and 21 kGy. Samples were evaluated for virus infectivity, PCR amplification of fragments of HAdV-5 genome and abundance and antigenicity of the virion structural proteins. The maximum reduction in viral titre, in plaque-forming units (PFU) per millilitre, was about 7 and 5 log PFU/mL for e-beam irradiation at 20 kGy in PBS and 19 kGy in wastewater, respectively. Among the virion structural proteins detected, the hexon protein showed the higher radioresistance. Long (10.1 kbp) genomic DNA fragments were differently PCR amplified, denoting a substrate effect on HAdV-5 genome degradation by e-beam. The differences observed between the two substrates can be explained by the protective effect that the organic matter present in the substrate may have on viral irradiation. According to the obtained results, the decrease in viral viability/infectivity may be due to DNA damage and to protein alterations. In summary, electron beam irradiation at a dose of 13 kGy is capable of reducing HAdV-5 viral titres by more than 99.99% (4 log PFU/mL) in both substrates assayed, indicating that this type of technology is effective for viral wastewater disinfection and may be used as a tertiary treatment in water treatment plants. KEY POINTS: • The substrate in which the virus is suspended has an impact on its sensitivity to e-beam treatment. • E-beam irradiation at 13 kGy is capable of reducing by 4 Log PFU/mL the HAdV-5 viral titre. • The decrease in viral viability/infectivity may be due to DNA damage and to protein alterations.

Lulworthia atlantica: a new species supported by molecular phylogeny and morphological analysis.

This study presents morphological and molecular evidence for the establishment of a new species of Lulworthia based on the evaluation of 11 isolates of Lulworthiales collected in two marinas on the west coast of Portugal initially classified as Lulworthia sp. Maximum likelihood and Bayesian methods were applied to obtain phylogenetic trees for 18S-28S and internal transcribed spacer (ITS1-5.8S-ITS2 = internal transcribed spacer [ITS]) nuc rDNA. The isolates consistently grouped together in a distinct and highly supported clade; all other Lulworthiales analyzed, including the available sequences of Lulworthia spp., segregated separately. These results were supported by common and distinctive morphological characters and enabled recognition and description of the new species Lulworthia atlantica. Two Portuguese isolates were confirmed as Lulworthia cf. purpurea, and two were confirmed as asexual morphs of Zalerion maritima.

New Reports on the Portuguese Endemic Species, Santolina impressa: Secretory Structures, Essential Oil Composition and Antiviral Activity.

Santolina impressa is an aromatic Asteraceae species endemic to Portugal, traditionally used for its anti-inflammatory properties. The aim of this study was to characterize S. impressa secretory structures, analyze the essential oil (EO) from the aerial organs, and evaluate its antiviral activity against herpes simplex viruses HSV-1 and HSV-2. Secretory structures were investigated by light and scanning microscopy, and the secretion was histochemically characterized. The EO from the aerial organs in full blooming was analyzed by gas chromatography with flame ionization detection and gas chromatography-mass spectrometry. Antiviral assays were performed by direct contact with viral suspensions (virucidal effect), and in infected Vero E6 cells, at different time periods during the viral replication cycle. Two types of secretory structures were described, biseriate glandular trichomes and secretory ducts, producing an oleoresin and a resin rich in flavonoids, respectively. Fifty compounds were identified in S. impressa EO, accounting for 87% of the total constituents. Monoterpenes constituted the main EO fraction (82%), with ß-pinene (13%) and ß-phellandrene (10%) being their major components. The EO interacted with HSV-1 and HSV-2 in a dose-dependent manner, thereby inactivating both viral infections. The EO did not evidence a virucidal effect but inhibited the HSV-1 and HSV-2 infection in Vero cells in a dose-dependent manner. However, further studies are needed to investigate the mode of action in the replication cycle.

Use of drift substrates to characterize marine fungal communities from the west coast of Portugal.

This survey reports the occurrence, diversity and similarity of marine fungi associated with five categories of drift substrates (Arundo donax, Phragmites australis, Spartina maritima, "other stems" and driftwood) collected on four sandy beaches of the western coast of Portugal. "Other stems" and driftwood are composite samples with a variety of identified and unidentified pieces of non-woody and woody substrates respectively. Fifty-six taxa were identified, including 38 Ascomycota and 18 anamorphic fungi. Twenty-six taxa were generalists; however several cases of "substrate recurrence" were identified. The very frequent fungi differed among the categories of studied substrates, with the exception of Corollospora maritima, very frequent on four categories. Except for S. maritima, P. australis and driftwood, cases of multiple fungal colonization were rare. S. maritima was the single substrate with five different marine fungi on one sample, as well as with the highest number of very frequent fungi, highest percentage of colonization and average number of fungi per sample. Driftwood presented the highest value of fungal richness (37 taxa) and A. donax the lowest (22 taxa). ANOSIM analysis of similarity showed that all substrates supported different fungal communities with the exception of the pair P. australis/"other stems". The effect of sample size on estimated fungal richness was tested, and the results let us conclude that, although most of the sporadic fungi (<1% occurrence) will be detected only in a very large number of samples, 60 samples of A. donax and "other stems" and 70 samples of all the other substrates may suffice to assess their respective representative marine mycota.

One-Year Surveillance of SARS-CoV-2 Virus in Natural and Drinking Water.

Although the SARS-CoV-2 virus has been detected in wastewater from several countries, monitoring its presence in other water matrices is still limited. This study aimed to evaluate the presence of this virus in natural and drinking water over one year of monitoring (2021). A survey of viral RNA was carried out by RT-qPCR in concentrated samples of surface water, groundwater, and drinking water from different regions of Portugal. SARS-CoV-2 RNA-quantified in genomic copies per liter (gc/L) of sampled water-was not detected in groundwater, but was detected and quantified in samples of surface water (two out of 43; 8035 and 23,757 gc/L) and in drinking water (one out of 43 samples; 7463 gc/L). The study also detected and quantified Norovirus RNA, intending to confirm the use of this enteric virus to assess variations in fecal matter throughout the sampling campaign. The samples positive for SARS-CoV-2 RNA also had the highest concentrations of Norovirus RNA-including the drinking water sample, which proved negative for fecal enteric bacteria (FIB). These results indicate that, to protect human health, it is advisable to continue monitoring these viruses, and noroviruses as fecal indicators (FI) as well-especially in low-flow water bodies that receive wastewater.

New viruses from Lacerta monticola (Serra da Estrela, Portugal): further evidence for a new group of nucleo-cytoplasmic large deoxyriboviruses.

Lizard erythrocytic viruses (LEVs) have previously been described in Lacerta monticola from Serra da Estrela, Portugal. Like other known erythrocytic viruses of heterothermic vertebrates, these viruses have never been adapted to cell cultures and remain uncharacterized at the molecular level. In this study, we made attempts to adapt the virus to cell cultures that resulted instead in the isolation of a previously undetected Ranavirus closely related to FV3. The Ranavirus was subsequently detected by polymerase chain reaction (PCR) in the blood of infected lizards using primers for a conserved portion of the Ranavirus major capsid protein gene. Electron microscopic study of the new Ranavirus disclosed, among other features, the presence of intranuclear viruses that may be related to an unrecognized intranuclear morphogenetic process. Attempts to detect by PCR a portion of the DNA polymerase gene of the LEV in infected lizard blood were successful. The recovered sequence had 65.2/69.4% nt/aa% homology with a previously detected sequence from a snake erythrocytic virus from Florida, which is ultrastructurally different from the studied LEV. These results further support the hypothesis that erythrocytic viruses are related to one another and may represent a new group of nucleo-cytoplasmic large deoxyriboviruses.

Assessment of the Presence of Hepatitis E virus in Surface Water and Drinking Water in Portugal.

Hepatitis E virus (HEV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepeviridae family, resistant to environmental conditions, and transmitted by the consumption of contaminated water. This virus is responsible for both sporadic and epidemic outbreaks, leading to thousands of infections per year in several countries, and is thus considered an emerging disease in Europe and Asia. This study refers to a survey in Portugal during 2019, targeting the detection and eventual quantification of enteric viruses in samples from surface and drinking water. Samples positive for HEV RNA were recurrently found by reverse transcription quantitative PCR (RT-qPCR), in both types of matrix. The infectivity of these samples was evaluated in cultured Vero E6 cells and RNA from putative viruses produced in cultures evidencing cytopathic effects and was subjected to RT-qPCR targeting HEV genomic RNA. Our results evidenced the existence of samples positive either for HEV RNA (77.8% in surface water and 66.7% in drinking water) or for infectious HEV (23.0% in surface water and 27.7% in drinking water). These results highlight the need for effective virological control of water for human consumption and activities.

Ocurrence of Ostreopsis in two temperate coastal bays (SW iberia): Insights from the plankton.

The benthic genus Ostreopsis contains toxic-bloom forming species and is an important cause of concern in warm-temperate and tropical waters. On the coast of Portugal, NE Atlantic, the occurrence of Ostreopsis cf. siamensis and Ostreopsis cf. ovata has been reported since 2008 and 2011, respectively. This work aims to understand the favorable conditions for high concentrations of Ostreopsis cells in the plankton at two sites, Lagos and Lisbon Bays, located in the South and West coast of Portugal, respectively. This study is based on weekly Ostreopsis abundance data in the plankton, from 2011 to 2017, daily satellite and in situ sea surface temperature (SST), and meteorological and sea state parameters, namely wind stress and significant wave height. The molecular identification of local Ostreopsis spp. is also presented. The maximum cell densities occur between late-summer and autumn. The distribution range of Ostreopsis cf. ovata is restricted to the South coast, while Ostreopsis cf. siamensis has a wider distribution range, being also present on the West coast. In the study period, there was only one occurrence of Ostreopsis spp., in Lagos Bay, with concentrations within the alert phase of monitoring. In Lagos Bay, high Ostreopsis spp. concentrations were related with positive SST anomalies. These high concentrations were often recorded after a period of almost 2-weeks to more than 4-weeks of low sea state (<0.6 m), followed by short time events of onshore wind and moderate waves (0.6-1 m). The former conditions are interpreted as favoring bloom development on the substrate and the latter as causing the re-suspension of Ostreopsis cells in the water column. In Lisbon Bay, O. cf. siamensis occurred in the plankton in few occasions and no clear relation could be established with the studied environmental variables. It is here hypothesized that the recent records of O. cf. siamensis in Lisbon Bay may correspond to an early colonization stage of an invasion process. Knowledge gained on Ostreopsis dynamics along the Portuguese coast can be used for both the improvement of benthic harmful algal blooms (BHABs) monitoring in the region and as a basis to design forecasting models.

Genetic Diversity in Natural Populations and Cultivated Stands of Walnut (Juglans regia L.) Using Microsatellite Marker

Abstract Information on genetic diversity is fundamental to developing in situ or ex situ conservation strategies. This study assessed the genetic differentiation between plantations and neighboring natural populations of Juglans regia. Genetic structures of three natural population and three neighboring plantations of J. regia in northwest of Iran were assessed using 10 nuclear microsatellite loci (SSR). Natural populations presented higher total number of alleles (119) and observed heterozygosity (Ho= 0.29) than planted stands (101 alleles, Ho= 0.21). The observed alleles of natural stands varied from 2 (WGA61 and WGA9) to 7 (WGA9) and from 2 (WGA321 and WGA276) to 5 (WGA202 and WGA9) in planted stands. One of the planted populations (B) indicated the largest level of genetic diversity. In conclusion, genetic diversity of all investigated plantation and natural stands are similar. This recommends that even plantations might qualify as gene conservation stands.