BACKGROUND: African swine fever (ASF) is a highly contagious and severe haemorrhagic disease of Suidae, with mortalities that approach 100 percent. Several studies suggested the potential implication of non-biting dipterans in the spread of ASFV in pig farms due to the identification of the ASFV DNA. However, to our knowledge, no study has evaluated the viral DNA load in non-biting dipterans collected in outbreak farms and no risk factors have been analysed. In this context, our study aimed to analyse the risk factors associated with the presence of non-biting dipterans collected from ASF outbreaks in relation to the presence and load of viral DNA.
METHODS: Backyard farms (BF), type A farms (TAF), and commercial farms (CF), were targeted for sampling in 2020. In 2021, no BF were sampled. Each farm was sampled only once. The identification of the collected flies to family, genus, or species level was performed based on morphological characteristics using specific keys and descriptions. Pools were made prior to DNA extraction. All extracted DNA was tested for the presence of the ASFV using a real-time PCR protocol. For this study, we considered every sample with a CT value of 40 as positive. The statistical analysis was performed using Epi Info 7 software (CDC, USA).
RESULTS: All collected non-biting flies belonged to five families: Calliphoridae, Sarcophagidae, Fanniidae, Drosophilidae, and Muscidae. Of the 361 pools, 201 were positive for the presence of ASFV DNA. The obtained CT values of the positive samples ranged from 21.54 to 39.63, with a median value of 33.59 and a mean value of 33.56. Significantly lower CT values (corresponding to higher viral DNA load) were obtained in Sarcophagidae, with a mean value of 32.56; a significantly higher number of positive pools were noticed in August, mean value = 33.12.
CONCLUSIONS: Our study brings compelling evidence of the presence of the most common synanthropic flies near domestic pig farms carrying ASFV DNA, highlighting the importance of strengthening the biosecurity measures and protocols for prevention of the insect life cycle and distribution.

Umbravirus-like associated RNAs (ulaRNAs) are a new group of subviral RNAs associated with plants. Little is known about the biology of ulaRNAs. We recently reported wheat umbra-like virus (WULV) from Kansas fields. In this work, we generated a full-length cDNA clone of WULV which systemically infected N. benthamiana. While agroinfiltrated leaves demonstrated severe necrosis, upper leaves were symptomless. We also showed that WULV is capable of infecting wheat in the absence of a helper virus. Furthermore, and through sap inoculation, we demonstrated that WULV is transmissible in the form of free RNA. This is the first report of a mechanically transmissible ulaRNA. Together, these findings contribute to advancing our knowledge of the biology of WULV. Moreover, the construction of the WULV infectious clone provides a valuable research tool for further investigations including the role of WULV in symptom development in interaction with other wheat viruses.

An analysis of the complete genome sequence of a novel isolate of tomato brown rugose fruit virus (ToBRFV) obtained from tomatoes in Iran and named ToBRFV-Ir is presented in this study. Comprehensive phylogenetic analysis utilizing key viral proteins, including 126 KDa, 183 KDa, movement protein (MP), and coat protein (CP), as well as the complete genome sequence, classified ToBRFV-Ir and 65 isolates from GenBank into three distinct clades. Notably, genetic diversity assessment revealed relatively low variability among the isolates, irrespective of their geographical or clade affiliation. Natural selection analysis based on the complete genome sequence showed that dN/dS values were consistently <1, indicating the prevailing role of negative selection across all populations. Analyses using the Recombination Detection Program and SplitsTree found no evidence of recombination events or signals in the complete genome sequence of the tested isolates. Thus, these results suggest that the genetic composition of ToBRFV remains stable without significant genetic exchange or recombination events occurring. A simple arithmetic comparison of the patristic distances and dates suggested that the time to the most recent common ancestor (TMRCA) of the ToBRFV populations is approximately 0.8 up to 2.7 with the closest tobamoviruses. An evolutionary study of the tested isolates from various countries based on the complete genome suggests Peruvian ancestry. The ToBRF-Ir isolate was successfully transmitted through mechanical inoculations to Solanum lycopersicum and Nicotiana rustica. These findings shed light on the genetic dynamics and transmission mechanisms of ToBRFV, providing valuable insights into its molecular characteristics and potential spread among susceptible plant species.

Oriental theileriosis, a disease primarily impacting cattle is caused by an apicomplexan hemoprotozoan parasite, Theileria orientalis. It has now become established in the Australasia region. The organism was long considered a benign cause of persistent infections; however, an increase in clinical outbreaks since 2006 in the eastern Australian states and New Zealand was associated with the identification of the pathogenic Ikeda (Type 2) and Chitose (Type 1) genotypes. Unlike the pathogenic T. parva and T. annulate, which target leucocytes, clinical manifestation with T. orientalis is due to its effects on erythrocytes, with the infection sometimes designated as Theileria associated bovine anemia (TABA). In Australia and New Zealand, the tick Haemaphysalis longicornis is the principal vector, though other Haemaphysalis species are also likely vectors. The endemic status of infection with pathogenic genotypes in areas with low or absent tick populations is an apparent paradox that may be attributable to alternative modes of transmission, such as mechanical transmission by hematophagous insects (lice, mosquitoes, and biting flies), vertical transmission, and transmission via iatrogenic means. This review addresses the evidence for the different modes of transmission of T. orientalis with particular focus on the reported and potential vectors in Australasia.

Trichosanthes kirilowii has been mainly grown for use in traditional Chinese medicine. In this study, cucurbit mild mosaic virus (CuMMV) belonging to the genus Fabavirus was identified from T. kirilowii plants. CuMMV possesses a segmented, bipartite linear single-stranded RNA genome composed of RNA1 and RNA2. Sequence analysis showed that each genomic segment shares the highest sequence similarity with those of CuMMV isolated from pumpkin. A full-length infectious cDNA clone of CuMMV was further constructed and was found to induce typical symptoms in T. kirilowii, Cucumis sativus, C. melo, Citrullus lanatus, and Cucurbita pepo. The sap inoculum derived from the infectious cDNA clone of CuMMV could be mechanically transmitted and reproduce similar symptoms in the tested plants. This is the first report on the construction of a biologically active, full-length infectious cDNA clone of CuMMV, which will provide a useful tool in understanding CuMMV-encoded proteins and plant-CuMMV interactions.

UNASSIGNED: Mosquitoes either biologically or mechanically transmit various vector-borne pathogens affecting pigs. Mosquito species display a wide variety of host preference, as well as host attraction and behaviours. Mosquito species attraction rates to- and feeding rates on pigs or other potential hosts, as well as the seasonal abundance of the mosquito species affects their pathogen transmission potential.
UNASSIGNED: We caught mosquitoes in experimental cages containing pigs situated in Romanian backyard farms. The host species of blood meals were identified with PCR and sequencing.
UNASSIGNED: High feeding preferences for pigs were observed in Aedes vexans (90%), Anopheles maculipennis (80%) and Culiseta annulata (72.7%). However, due to a high abundance in the traps, Culex pipiens/torrentium were responsible for 37.9% of all mosquito bites on pigs in the Romanian backyards, despite low feeding rates on pigs in the cages (18.6%). We also found that other predominantly ornithophilic mosquito species, as well as mosquitoes that are already carrying a blood meal from a different (mammalian) host, were attracted to backyard pigs or their enclosure.
UNASSIGNED: These results indicate that viraemic blood carrying, for instance, African swine fever virus, West-Nile virus or Japanese encephalitis virus could be introduced to these backyard pig farms and therefore cause an infection, either through subsequent feeding, via ingestion by the pig or by environmental contamination.

Parapoxvirus (PPV) causes papular stomatitis and contagious pustular dermatitis in ruminants worldwide. The virus is generally transmitted through close contact with skin lesions containing PPV in infected animals and indirectly through PPV-contaminated materials. PPV-infected animals frequently do not show clinical signs and the route of PPV transmission is sometimes unclear. In this study, the possibility of mechanical transmission of PPV by houseflies (Musca domestica) was investigated using polymerase chain reaction (PCR) gene surveillance. Samples were collected from cattle, sheep, barn environments, direct wash solution of the body surface of houseflies, and indirect wash solution of the body surface and feces of the flies. Bovine papular stomatitis virus, pseudocowpox virus, and orf virus were detected in the oral cavity and body surface of cattle and sheep without clinical signs of PPV infection or barn environments; PPV was considered to have been retained on the farm. PPVs were also detected in the direct wash solution of the body surface of houseflies, and the indirect wash solution of the body surface and feces of the flies. The viral sequence determined from the indirect wash solution of the body surface and feces of the flies was identical to that determined from the body surface of cattle and barns. These results suggested that houseflies may mechanically transmit PPV to both cattle and sheep.

Trichosanthes kirilowii is one of the most important perennial herbaceous vines that have been used in traditional Chinese medicine. In this study, a novel RNA virus was discovered in T. kirilowii plants showing leaf mottling and mosaic symptoms. The complete genome of this virus is 6,524 nucleotides long and encodes four open reading frames which are arranged in a manner typical of tobamoviruses. Phylogenetic analysis based on the complete genome sequence revealed that the virus was clustered into a branch with the tobamoviruses whose natural host are plants belonging to the family Cucurbitaceae. A full-length infectious cDNA clone was then constructed and demonstrated to establish a systemic infection with typical symptoms in Nicotiana benthamiana, T. kirilowii, and five other cucurbitaceous crops including Cucumis melo, C. lanatus, C. sativus, Luffa aegyptiaca, and Cucurbita pepo via agrobacterium-mediated infectivity assays. Further experiments provided evidence that the rod-shaped viral particles derived from the infectious clone could be mechanically transmitted and reproduce indistinguishable symptoms in the tested plants. Taken together, the mottle mosaic disease of T. kirilowii is caused by a distinct tobamovirus, for which the name Trichosanthes mottle mosaic virus (TrMMV) is proposed. As the infectious cDNA clone of TrMMV could also infect five other cucurbit crops, this distinct tobamovirus could be a potential threat to other cucurbitaceous crops.

Flies and other arthropods mechanically transmit multiple pathogens and a recent experimental study demonstrated house flies, Musca domestica L. (Diptera: Muscidae), can mechanically transmit SARS-CoV-2. The purpose of this study was to explore the possibility of mechanical transmission of SARS-CoV-2 by domestic insects and their potential as a xenosurveillance tool for detection of the virus. Flies were trapped in homes where at least one confirmed human COVID-19 case(s) resided using sticky and liquid-baited fly traps placed inside and outside the home in the Texas counties of Brazos, Bell, and Montgomery, from June to September 2020. Flies from sticky traps were identified, pooled by taxa, homogenized, and tested for the presence of SARS-CoV-2 RNA using quantitative reverse transcription PCR (RT-qPCR). Liquid traps were drained, and the collected fluid similarly tested after RNA concentration. We processed the contents of 133 insect traps from 40 homes, which contained over 1,345 individual insects of 11 different Diptera families and Blattodea. These individuals were grouped into 243 pools, and all tested negative for SARS-CoV-2 RNA. Fourteen traps in seven homes were deployed on the day that cat or dog samples tested positive for SARS-CoV-2 RNA by nasal, oral, body, or rectal samples. This study presents evidence that biting and nonbiting flies and cockroaches (Blattodea) are not likely to contribute to mechanical transmission of SARS-CoV-2 or be useful in xenosurveillance for SARS-CoV-2.

The European Commission requested that EFSA provide study designs for the investigation of four research domains according to major gaps in knowledge identified by EFSA in a report published in 2019: (i) the patterns of seasonality of African Swine Fever (ASF) in wild boar and domestic pigs in the EU; (ii) the epidemiology of ASF in wild boar; (iii) survival of ASF virus (ASFV) in the environment and (iv) transmission of ASFV by vectors. In this Scientific Opinion, the fourth research domain on ASFV transmission by vectors is addressed. Eleven research objectives were proposed by the EFSA working group and broader ASF expert networks, such as ASF stop, ENETWILD, VectorNet, AHAW network and the AHAW Panel Experts. Of the 11 research objectives, six were prioritised based on the following set of criteria: (1) the impact on ASF management; (2) the feasibility or practicality to carry out the study; (3) the potential implementation of study results in practice; (4) a possible short time-frame study (< 1 year); (5) the novelty of the study and (6) if it was a priority for risk managers. The prioritised research objectives were: (I) Studies on the potential vector fauna at the pig-wild boar interface and the feeding preference of blood-feeding potential vectors in ASF-affected areas; (II) Assessment of the efficacy of insect screens on indoor/outdoor pig holdings to prevent the entry of blood-sucking vectors (i.e. Stomoxys) in ASF endemic areas; (III) Assess the role of mechanical vectors in the virus transmission in ASF-affected areas; (IV) Distribution of the potential mechanical transmission vectors in ASF-affected areas of the EU; (V) ASFV transmission by synanthropic birds; and (VI) Assessment on the presence/absence of the soft tick Ornithodoros erraticus in ASF-affected areas in Europe. For each of the selected research objectives, a research protocol has been proposed considering the potential impact on ASF management and the period of 1 year for the research activities.