Tenacity of Animal Disease Viruses on Wood Surfaces Relevant to Animal Husbandry.

The aim of this study was to analyse the hygienic suitability of wood often used in animal husbandry. To this end, the inactivation of viruses (Enterovirus E as a surrogate for non-enveloped viruses and Newcastle disease virus as a surrogate for enveloped viruses) on germ carriers consisting of various types of wood was studied over an extended period to assess the biosafety of wood as an agricultural building material. The study was designed to assess the intrinsic biocidal activity of the wood itself, without the use of a disinfectant. The laboratory tests were based on German test guidelines and current European standards. Five different types of wood germ carriers, i.e., spruce (Picea abies), pine (Pinus sylvestris), poplar (Populus sp.), beech (Fagus sylvatica) and Douglas fir (Pseudotsuga menziesii), as well as stainless-steel carriers, were inoculated with enveloped and non-enveloped viruses and stored for up to four months, and the remaining infectivity of the viruses was continuously assessed. The results showed that intact, finely sawn timber with a low depth of roughness had an inactivating effect on the viruses up to 7.5 decadal logarithmic levels. For the non-enveloped virus, inactivation was fastest on Douglas fir wood, with the target reduction for effective inactivation (reduction by factor 4.0 log10) being achieved after two weeks, and for the enveloped virus on pine wood, it was already achieved from the day of drying. The hygienic effects of the wood carriers may be due to their hygroscopic properties and wood constituents. These effects offer potential for further investigation, including tests with other wood species rich in extractives.

SARS-CoV-2 RNA Detection on Disposable Wooden Chopsticks, Hong Kong.

We detected severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA on disposable wooden chopsticks used by 5 consecutive asymptomatic and postsymptomatic patients admitted for isolation and care at our hospital. Although we did not assess virus viability, our findings may suggest potential for transmission through shared eating utensils.

Role of Fresh Dead Wood in the Epidemiology and the Biological Control of the Chestnut Blight Fungus.

The invasive fungus Cryphonectria parasitica, the causal agent of chestnut blight, is able to survive and sporulate on the bark of fresh dead Castanea sativa wood for at least 2 years. Here, we experimentally investigated the role of fresh dead wood in the epidemiology of chestnut blight, specifically in the spread of the hyperparasitic virus Cryphonectria hypovirus 1, which acts as biocontrol agent of C. parasitica. A total of 152 artificially initiated, virulent bark cankers in four chestnut stands were treated with virus-infected asexual spores originating either from sporulating dead wood or from a spore suspension. Molecular markers for both the virus and the fungal carrier were used to examine the spread of the applied biocontrol virus. Fourteen months after treatment, 42 to 76% of the conidial spray-treated cankers and 50 to 60% of the cankers exposed to a sporulating dead stem had been virus infected by the applied hypovirulent conidia in all four study sites. Virus infection reduced canker expansion and promoted canker healing (callusing). Thus, fresh chestnut dead wood may play an important role in supporting the successful spread of natural hypovirulence in chestnut forests. Further, combined with the application of virus-infected conidial suspensions, it may help promote the establishment of artificially released hypoviruses in chestnut stands to control chestnut blight.

Comparison of Swab Sampling Methods for Norovirus Recovery on Surfaces.

Human noroviruses (HuNoVs) can be easily transferred by the contacts of humans or fomites. Swab sampling methods are widely used for recovering HuNoVs from small surfaces of various fomites or hard-to-reach locations and swab sampling conditions are important for the accurate detection of HuNoVs, which have a low infectious dose and relatively long persistence under a range of environmental conditions. Therefore, to determine the suitable swab sampling method for recovering HuNoVs from various surfaces, we evaluated combinations of four swab materials (cotton, microdenier polyester [a type of microfiber], polyurethane foam, and rayon) and three elution buffer solutions (phosphate-buffered saline [PBS], PBS with 0.2% Tween-80, and 3% beef extract-50 mM glycine [pH 9.5]). First, we inoculated HuNoVs or murine noroviruses (MuNoVs), the surrogate of HuNoVs, onto test coupons (10 × 10 cm) consisting of three common surface materials (high-density polyethylene, stainless steel, and wood). Coupons were swabbed using a combination of each swab material and elution buffer, and the viral recovery was measured by real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) or plaque assay. By RT-qPCR, we confirmed that the cotton swab-PBS and microdenier polyester-PBS combinations had recovery efficiencies greater than 80% for viruses on plastic and stainless steel surfaces. The cotton swab-PBS combination had the highest recovery efficiency on all surface materials via the plaque assay. Therefore, a cotton or a microdenier polyester swab with PBS could be a useful method for sampling HuNoVs on various surfaces.

Use of siRNAs for Diagnosis of Viruses Associated to Woody Plants in Nurseries and Stock Collections.

Woody perennial plants like grapevine and fruit trees can be infected by several viruses even as multiple infections. Since they are propagated vegetatively, the phytosanitary status of the propagation material (both the rootstock and the variety) can have a profound effect on the lifetime and health of the new plantations. The fast evolution of sequencing techniques provides a new opportunity for metagenomics-based viral diagnostics. Viral derived small RNAs produced by the host immune system during viral infection can be sequenced by next-generation techniques and analyzed for the presence of viruses, revealing the presence of all known viral pathogens in the sample. This method is based on Illumina sequencing of short RNAs and bioinformatics analysis of virus-derived small RNAs in the host. Here we describe a protocol for this challenging technique step by step with notes, in order to ensure success for every user.

Discovery of Negative-Sense RNA Viruses in Trees Infected with Apple Rubbery Wood Disease by Next-Generation Sequencing.

Apple rubbery wood is a disease of apple found around the world, often associated with Apple flat limb disease, and regulated in many countries. Despite its long history in apple cultivation, the disease's causal agent has remained elusive. In this study, next-generation sequencing (NGS) was used to identify and characterize several related novel viral agents from apple rubbery wood-infected plants, which have been named Apple rubbery wood virus (ARWV) 1 and 2. Additional specimens with apple rubbery wood disease tested positive by polymerase chain reaction with primers designed to ARWV 1 and 2 genomic RNA segments. In an NGS-based screening of over 100 Malus and 100 Prunus specimens from a collection of virus-infected trees, only one Malus specimen was found to be infected with ARWV not known to be infected with the disease, which strongly suggests that ARWV is not commonly found in Malus spp. or other fruit trees. The two viruses are most closely related to members of the order Bunyavirales. Three RNA segments (large, medium, and small) were characterized and the viruses likely represent a new genus under the family Phenuiviridae, with a suggested name of Rubodvirus (Rubbery wood virus).

A simplified strategy for studying the etiology of viral diseases: Apple stem grooving virus as a case study.

A simple method to amplify infective, complete genomes of single stranded RNA viruses by long distance PCR (LD PCR) from woody plant tissues is described in detail. The present protocol eliminates partial purification of viral particles and the amplification is achieved in three steps: (i) easy preparation of template RNA by incorporating a pre processing step before loading onto the column (ii) reverse transcription by AMV or Superscript reverse transcriptase and (iii) amplification of cDNA by LD PCR using LA or Protoscript Taq DNA polymerase. Incorporation of a preprocessing step helped to isolate consistent quality RNA from recalcitrant woody tissues such as apple, which was critical for efficient amplification of the complete genomes of Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV) and Apple chlorotic leaf spot virus (ACLSV). Complete genome of ASGV was cloned under T7 RNA polymerase promoter and was confirmed to be infectious through transcript inoculation producing symptoms similar to the wild type virus. This is the first report for the largest RNA virus genome amplified by PCR from total nucleic acid extracts of woody plant tissues.

Survival of norovirus surrogate on various food-contact surfaces.

Norovirus (NoV) is an environmental threat to humans, which spreads easily from one infected person to another, causing foodborne and waterborne diseases. Therefore, precautions against NoV infection are important in the preparation of food. The aim of this study was to investigate the survival of murine norovirus (MNV), as a NoV surrogate, on six different food-contact surfaces: ceramic, wood, rubber, glass, stainless steel, and plastic. We inoculated 10(5) PFU of MNV onto the six different surface coupons that were then kept at room temperature for 28 days. On the food-contact surfaces, the greatest reduction in MNV was 2.28 log10 PFU/coupon, observed on stainless steel, while the lowest MNV reduction was 1.29 log10 PFU/coupon, observed on wood. The rank order of MNV reduction, from highest to lowest, was stainless steel, plastic, rubber, glass, ceramic, and wood. The values of d R (time required to reduce the virus by 90%) on survival plots of MNV determined by a modified Weibull model were 277.60 h (R(2) = 0.99) on ceramic, 492.59 h (R(2) = 0.98) on wood, 173.56 h on rubber (R(2) = 0.98), 97.18 h (R(2) = 0.94) on glass, 91.76 h (R(2) = 0.97) on stainless steel, and 137.74 h (R(2) = 0.97) on plastic. The infectivity of MNV on all food-contact surfaces remained after 28 days. These results show that MNV persists in an infective state on various food-contact surfaces for long periods. This study may provide valuable information for the control of NoV on various food-contact surfaces, in order to prevent foodborne disease.

Disinfection of foot-and-mouth disease and African swine fever viruses with citric acid and sodium hypochlorite on birch wood carriers.

Transboundary animal disease viruses such as foot-and-mouth disease virus (FMDV) and African swine fever virus (ASFV) are highly contagious and cause severe morbidity and mortality in livestock. Proper disinfection during an outbreak can help prevent virus spread and will shorten the time for contaminated agriculture facilities to return to food production. Wood surfaces are prevalent at these locations, but there is no standardized method for porous surface disinfection; commercial disinfectants are only certified for use on hard, nonporous surfaces. To model porous surface disinfection in the laboratory, FMDV and ASFV stocks were dried on wood coupons and exposed to citric acid or sodium hypochlorite. We found that 2% citric acid was effective at inactivating both viruses dried on a wood surface by 30 min at 22°C. While 2000 ppm sodium hypochlorite was capable of inactivating ASFV on wood under these conditions, this chemical did not meet the 4-log disinfection threshold for FMDV. Taken together, our data supports the use of chemical disinfectants containing at least 2% citric acid for porous surface disinfection of FMDV and ASFV.

Cytomegalovirus survival on common environmental surfaces: opportunities for viral transmission.

Congenital cytomegalovirus (CMV) affects ~1 of 150 births and is a leading cause of hearing loss and intellectual disability. It has been suggested that transmission may occur via contaminated surfaces. CMV AD169 in filtered human saliva, applied to environmental surfaces, was recovered at various time points. Samples were evaluated by culture and real-time polymerase chain reaction. CMV was found viable on metal and wood to 1 hour, glass and plastic to 3 hours, and rubber, cloth, and cracker to 6 hours. CMV was cultured from 83 of 90 wet and 5 of 40 dry surfaces. CMV was more likely to be isolated from wet, highly absorbent surfaces at earlier time points.

The survival of avian reoviruses on materials associated with the poultry house environment.

Two strains of avian reovirus were tested for their ability to survive on materials common to most poultry houses. The viruses survived longest and for at least 10 days on feathers, wood shavings and chicken feed, and for the shortest periods on wood (2 days), paper and cotton (4 days). There were some differences in survivability between the two strains. In most instances, the presence of faecal material increased the survival time, although in others it had the opposite effect. Reovirus survived for at least 10 days on the surface of eggshells when organic material was present. In drinking water, it survived for at least 10 weeks with little loss of infectivity. This could have implications for contamination of water supplies in poultry houses. It was shown that if cotton swabs are used for sampling, reovirus survives longer if they are pre-moistened with culture medium rather than used dry.

[RT--PCR analysis of Phanerochaete chrysosporium lip genes in colonized fir wood].

Expression of Phanerochaete chrysosporium lip A2 (GLG3) lip C1 (GLG2) lip C2 (GLGS) lipD2( GLG1), lipE (LP0811) genes were analyzed by RT-PCR method. It was showed that some genes were expressed in special colonized period. Only lip A2 (GLG3) transcription occured in the 2nd week and the 8th week, both lip C1 (GLG2) and lip D2 (GLG1) gene transcription was checked out in the period of 6 weeks. However, no lip genes were expressed in the time of 4 weeks. These results indicated that lip gene expression is relied on the colonized period and transcript patterns are re dramatically different from those in previous studies with defined media.