Laparoscopic surgery has become the preferred surgical approach of several colorectal conditions. However, the economic results of this are quite controversial. The degree of adoption of laparoscopic technology, as well as the aptitude of the surgeons, can have an influence not only in the clinical outcomes but also in the total procedure cost. The aim of this study was to evaluate the clinical and economic outcomes of laparoscopic colorectal surgeries, compared to open procedures in Brazil.All patients who underwent elective colorectal surgeries between January 2012 and December 2013 were eligible to the retrospective cohort. The considered follow-up period was within 30 days from the index procedure. The outcomes evaluated were the length of stay, blood transfusion, intensive care unit admission, in-hospital mortality, use of antibiotics, the development of anastomotic leakage, readmission, and the total hospital costs including re-admissions.P < .001). There were no significant differences in other clinical outcomes between the 2 groups. The total costs were similar between the 2 groups, in the multivariate analysis . The cost predictors were the cancer diagnosis and age.Two hundred eighty patients, who met the eligibility criteria, were included in the analysis. Patients in the laparoscopic group had a shorter length of stay in comparison with the open group (6.02 ± 3.86 vs 9.86 ± 16.27, Laparoscopic colorectal surgery presents a 17% decrease in the duration of the hospital stay without increasing the total hospitalization costs. The factors associated with increased hospital costs were age and the diagnosis of cancer. Many studies, including randomized clinical trials, have shown that laparoscopic access is both effective and safe, with better short-term results in patient recovery compared to the open surgical approach.,9,14–18Regarding long-term clinical outcomes, studies have shown that there are no differences between laparoscopic or open surgery in cancer patients, or that laparoscopic surgery is superior to open surgery in the disease-free survival rate of colonic resections.–22 others have demonstrated that laparoscopy may be either cost-neutral or yield savings from the perspective of hospitals and the payers.,11,23–26Regarding the economic outcomes, the evidence is rather controversial. Although a few publications have shown that laparoscopic colorectal surgery is more costly, despite the better short-term clinical outcomes, Besides, laparoscopy adoption in a country, as well as the degree of aptitude of the surgeons can influence the clinical outcomes and, consequently, the total procedure cost.,28–30 Some publications have shown that after the learning curve period, laparoscopic surgery may either be cost-neutral, or cost-saving compared to the open surgical approach.The difference in the results might be explained by the impact of the cost of the device in the total hospital cost, according to the healthcare system and health technology incorporation characteristics of each country.Thus, the objective of this study was to evaluate the clinical and economic outcomes of laparoscopic colorectal surgeries, compared to open procedures, in the Brazilian private healthcare system according to the perspective of the payers and providers.22.1The study was a retrospective cohort. The dataset used for the analyses was a hospital patients’ billings database (reimbursement information) from Orizon. The database contains information about the transaction between health insurance plans and hospitals from the Brazilian private healthcare system, including all the billed items during hospital admissions. It also contains information from ambulatory service transactions. About 12 million patients – who represent around 25% of patients in the Brazilian private healthcare system – are included in Orizon's registries.All patients who were admitted between January 2012 and December 2013 who underwent rectum or sigmoid colon procedures  were considered potentially eligible. The TUSS codes used to determine the procedures were: 31003559 – retossigmoidectomia abdominal  and 31003796 – retossigmoidectomia abdominal por videolaparoscopia . The eligible patients had their procedure in multiple location across different states in Brazil, but due to confidentiality rules, the location of the patients was not described in the dataset.The following eligibility criteria were used for the inclusion of patients:Availability of age, gender, international classification of diseases (ICD) code information and surgical approach (laparoscopic vs open);The procedure should be elective (urgent surgeries were excluded).The colorectal surgery must have been performed in a maximum of 3 days after hospital admission;The outcomes of perioperative procedures and readmission for up to 30 days were analyzed. As all the analyzed data were obtained from the administrative claims data of the routine care of the patients and were made anonymous by the researchers, no informed consent was required. Lastly, as there was no possibility of any harm arising as a result of the conduct of this research, no Ethical Review Board approval was required.2.2The Patients were grouped according to surgical approach (laparoscopic vs open). The following variables were evaluated for each patient: age, gender, ICD code for the admission related to the surgery: cancer status, use of chemotherapy and radiotherapy, extent of the surgery , need for perioperative blood transfusion, length of stay (LOS), intensive care unit (ICU) admission, 30-day readmission, the use of antibiotics, mortality, the occurrence of anastomotic leakage, and hospital costs.Surgical treatment involving only the rectum or sigmoid colon with or without lymph nodes resection and with or without colostomy associated, were classified as simple procedures. If an additional procedure including hysterectomy, oophorectomy, or cystectomy were billed on the same day of the colorectal procedure, the surgery was classified as a multivisceral resection.Perioperative transfusion was defined as the use of a concentrate of red blood cells between the date of surgery and the following 30 days. Readmissions were defined whenever a patient had billed items for at least 2 consecutive days. Only an emergency room visit was not considered readmission.rd and 4th generation cephalosporins, vancomycin, aminoglycosides, piperacillin, and a combination of quinolones with either clindamycin or metronidazole.The rate of the use of postoperative antibiotics was defined as a postoperative exposure (up to 30 days from index surgery) to antimicrobial drugs, which suggested a therapeutic use – whenever the utilization was suggestive of prophylaxis, it was not considered. The criteria used were: prescriptions for at least 7 days and use of 1 of the following antimicrobials: carbapenems, 3The definition of anastomotic Leakage occurrence was the result of the simultaneous presence of the following conditions:1.The patient had an infection under investigation, where the proxy used was the billing of a postoperative blood culture exam;2.There was at least 1 postoperative imaging study, such as an abdominal X-ray, computerized tomography or another abdominal exam with an intravenous contrast.3.A second surgical abdominal procedure or image-guided percutaneous drainage must have been undertaken up to 30 days after the index surgery.Hospital costs were categorized as index admission costs, which included all billed items during the index surgery admission, and 30-day readmissions costs, which included all the billed items from any other hospital admission initiated within 30 days of the index surgery. In both cases, we did not use a termination date. If the index admission or a readmission began in less than 30 days after the index surgery and lasted for example 4 months, all expenditures associated with this admission were included. The total patients’ costs were also calculated - by summing the costs of the index admission and 30-day readmission.2.3t test for continuous variables with normal distribution. Considering that both costs and LOS, the 2 main continuous outcomes of this study, usually present a gamma distribution, we also employed univariate generalized linear model (GLM) with the gamma distribution for these analyses.Categorical variables are presented as absolute values and proportions, and continuous variables are presented as the mean and standard deviation. Comparison between the laparoscopic versus open surgery groups employed the Student Fisher exact test for categorical variables and the Student P value of .10 or less in the univariate analyses were selected to enter the multivariate analyses. In all analyses, a P value smaller than .05 was considered statistically significant. SPSS  was used for the analyses.Multivariate analyses used Poisson regression for dichotomous outcomes  and GLMs for costs and LOS. Variables with a 3P = .001), and had a smaller proportion of cancer diagnosis , as shown in Table Among 1.321 potentially eligible patients, 280 fulfilled the eligibility criteria and were included in the analyses .The proportion of male patients and multivisceral resections did not show a statistically significant difference. Although the comparison of the 2 groups showed a higher proportion of patients receiving chemotherapy in the open surgery group, the proportion of such patients considering only the ones with cancer in each group did not reach statistically significant difference  in the analysis adjusted for age, gender, cancer status, and multivisceral resection. The incidence of anastomotic leak, readmissions, use of antibiotics and the mortality rate did not differ between the 2 groups.P = .024), as well as total costs, where 30-day readmissions were also computed .The mean index admission costs (presented in Table P = .074). Results for the index admission costs were very similar (data not being shown). The predictors that remained statistically significant in the multivariate analyses were the status of cancer  and the age .Conversely, in the GLM multivariate analysis of total costs Table , with ad4–14,21,24In this study, which compared economic and clinical outcomes between laparoscopic and open colorectal surgery in a sample of patients from the Brazilian private healthcare system, the main difference between the 2 groups was the LOS. After adjustment for age, gender, cancer status, and multivisceral resection in the multivariate analysis, the LOS was 17% shorter (95% CI: 2%–30%) in the laparoscopic group. This result is comparable to those found in the literature that show that laparoscopic rectal surgeries present a shorter hospitalization period compared to open surgeries.P = .069). The ICU admission rates, which were reduced by 34% in the univariate analysis, lost its significance in the analysis adjusted for age . It is likely that the analysis of clinical outcomes suffered from insufficient statistical power, especially for mortality, where the number of events was small.Regarding the other clinical outcomes in the current study, patients had a lower need of blood transfusion and a smaller ICU admission rate in the univariate analysis. However, these associations did not maintain statistical significance in the multivariate analysis. The requirement for blood transfusions, which showed a 53% decrease in univariate analysis (95% CI: 11%–75%), lost it significance in the analysis adjusted for age, gender, and the cancer status . However, this comparison lost statistical significance in the multivariate GLM. Age and cancer diagnosis were identified as independent risk factors to increase the total hospital costs in colorectal procedures  and , respectively.The results regarding index admission and total expenses (which included 30-day readmissions) demonstrated that the laparoscopic approach is not more costly than open surgery. In fact, in the univariate analysis, laparoscopic surgery was associated with a lower cost when compared to the open approach and presented a lower standard deviation, which could suggest that laparoscopy has a greater predictability of total costs in comparison with the open surgery . However, non-surgical costs, including laboratory, radiology, and nursing post-operative care were lower in the laparoscopic group. The total costs were 7% lower in the laparoscopic group, although this difference was not significant.In a study with rectal cancer patients, which used propensity score matching, laparoscopy had intraoperative costs which were 21% higher than the open approach  can lead to selection bias and compromise the generalizability of the study. In this regard, the high proportion of patients with an endometriosis ICD code is noteworthy. Although this disease is seldom presented as a frequent motive for colorectal surgeries, its rate can be substantial, as seen in a national colorectal surgeries database from France.Lastly, some of the 95% CI from the estimates were substantially wide (except in the costs GLM), suggesting that a larger sample would be important to confirm some of our findings.5The Real-World Evidence of the Brazilian Healthcare System shows that laparoscopic colorectal surgery presents a 17% decrease in the hospital stay time without increasing the total hospitalization costs. Factors associated with increased hospital costs were age and the diagnosis of cancer. These findings suggest that the laparoscopic procedure may be considered the preferred procedure in patients submitted to colorectal surgery, regardless of the type of pathology, whenever there are no contraindications. The study also concludes that the laparoscopic method can be economically sustainable even in developing countries that present budgetary constraints.All authors had full access to all the data in the study and assumed responsibility for all aspects of the work, ensuring that questions related to the accuracy or integrity of any part of the work were appropriately investigated and resolved. Furthermore, all authors were accountable for the parts of the work they did, able to identify which co-authors were responsible for specific other parts of the work and confident in the integrity of the contributions of their co-authors. Ulysses Ribeiro Jr affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned  have been explained. Ulysses Ribeiro Jr, Daiane Oliveira Tayar, Rodrigo Antonini Ribeiro, Priscila Andrade, and Silvio Mauro Junqueira Jr had substantial contribution to the conception and design of the work; Ulysses Ribeiro Jr, Daiane Oliveira Tayar, and Rodrigo Antonini Ribeiro had substantial contribution in the data acquisition and/or analysis and interpretation of the data of the work; Daiane Oliveira Tayar and Rodrigo Antonini Ribeiro drafted the manuscript; Ulysses Ribeiro Jr, Daiane Oliveira Tayar, Priscila Andrade, and Silvio Mauro Junqueira Jr reviewed the article critically for important intellectual content; all authors provided the final approval of the version to be published.Conceptualization: Ulysses Ribeiro Jr, Daiane Oliveira Tayar.Data curation: Ulysses Ribeiro Jr, Daiane Oliveira Tayar, Rodrigo Antonini Ribeiro.Formal analysis: Rodrigo Antonini Ribeiro.Funding acquisition: Priscila Andrade, Silvio Mauro Junqueira Jr.Investigation: Ulysses Ribeiro Jr.Methodology: Ulysses Ribeiro Jr, Daiane Oliveira Tayar, Rodrigo Antonini Ribeiro, Priscila Andrade, Silvio Mauro Junqueira Jr.Project administration: Priscila Andrade, Silvio Mauro Junqueira Jr.Resources: Priscila Andrade, Silvio Mauro Junqueira Jr.Software: Rodrigo Antonini Ribeiro.Supervision: Ulysses Ribeiro Jr, Daiane Oliveira Tayar, Priscila Andrade, Silvio Mauro Junqueira Jr.Validation: Ulysses Ribeiro Jr, Daiane Oliveira Tayar, Rodrigo Antonini Ribeiro, Priscila Andrade, Silvio Mauro Junqueira Jr.Visualization: Ulysses Ribeiro Jr, Daiane Oliveira Tayar, Rodrigo Antonini Ribeiro, Priscila Andrade, Silvio Mauro Junqueira Jr.Writing – original draft: Daiane Oliveira Tayar, Rodrigo Antonini Ribeiro.Writing – review & editing: Ulysses Ribeiro Jr, Daiane Oliveira Tayar, Rodrigo Antonini Ribeiro, Priscila Andrade, Silvio Mauro Junqueira Jr.


Vitivirus in the family Betaflexiviridae includes eleven viruses known to infect grapevine: grapevine vitiviruses A, B, D, E, F, G, H, I, J, L and M (GVA-GVM). Three of these viruses, GVA, GVB and GVD, have been associated with the etiology of rugose wood disease in grapevine and cause agronomically significant losses. The other vitiviruses were more recently discovered and their effects on grapevine are undetermined. To certify grape material for propagation as virus tested, an updated reverse transcription PCR (RT-PCR) assay to detect all known vitiviruses is desirable. To accomplish this, multiple grapevine vitivirus sequences were aligned at the amino acid level to search for conserved motifs. Two highly conserved motifs were found at an ideal distance for RT-PCR detection in the RNA-dependent RNA polymerase region of the replicase protein. The amino acid motifs were back translated to create degenerate primers and used to successfully amplify all eleven grapevine vitiviruses. The RT-PCR primers were used to test a panel of vitivirus-infected vines for inclusivity as well as vines infected with closely related viruses in the Betaflexiviridae family (i.e. grapevine pinot gris virus and grapevine rupestris stem pitting-associated virus) for exclusivity. Broader use of these primers to detect vitiviruses in other plant hosts was investigated. In summary, an end-point RT-PCR assay that detects all the known grapevine vitiviruses and potentially other members of the genus Vitivirus has been developed. The universal assay represents an alternative to individual assays to reduce the work associated with the diagnosis of vitiviruses, including for regulatory purposes.The genus  Vitivirus (family Betaflexiviridae) was created in 1997 for the classification of type member grapevine virus A (GVA), a plant virus discovered in grapevine with a filamentous flexuous particle differing from trichoviruses (genus Trichovirus) in its genomic arrangement [https://talk.ictvonline.org/taxonomy/), nine species of vitivirus infecting grapevine are recognized: Grapevine virus A, Grapevine virus B, Grapevine virus D, Grapevine virus E, Grapevine virus F, Grapevine virus G, Grapevine virus H, Grapevine virus I and Grapevine virus J. Since 2019, two more proposed vitiviruses were discovered in grapevine. Grapevine virus L (GVL) was initially identified in RNAseq data and later detected in multiple plants in Croatia, New Zealand and the United States [The genus d States .Three different vitiviruses have been associated with the etiology of rugose wood disease in grapevine, a disease with world-wide distribution . GVA is Reliable diagnostic methods are critical in determining the viral infection status of a grapevine. Multiple tests are available for the detection of vitiviruses, including biological indexing, real-time or end-point reverse transcription PCR (RT-PCR) and HTS. Biological indicators do not show symptoms for all vitiviruses infecting grape, and RT-PCR assays can fail to detect vitivirus variants containing nucleotide differences at critical primer binding locations. HTS is the most effective means of detecting all vitiviruses but its high cost at large scale limits its use as a screening tool. HTS data is helpful to inform and update RT-PCR primer design as new virus strains are continually being characterized.In this study, a universal end-point RT-PCR assay involving degenerate primers with the capacity of detecting all the known grapevine vitiviruses was developed. To validate the new assay, eleven grapevines each infected with one of the vitiviruses (i.e. GVA to GVM) were tested. Moreover, a field survey was conducted of known vitivirus-infected grapevines. Following the first reports of vitiviruses in grapevine, several vitiviruses have been discovered in other hosts ; consequ5’-GGNGARTTYGGNACNTTYTTYTTY-3’) and Vitivirus-RdRp-R (5’-NCKCCANCCRCARAANARNGG-3’) were generated, which amplify a 219-bp product.With the aim of designing a generic RT-PCR assay for all the known grapevine vitiviruses, all the currently available complete and near-complete genome sequences in GenBank were analyzed. Likewise, available amino acid (aa) sequences for the REP and CP of different grapevine vitiviruses were obtained from GenBank. Thus, similar sequences  were aligned using MUSCLE , and GenTrichovirus) and grapevine rupestris stem pitting-associated virus  were also analyzed to test for assay exclusivity. Like vitiviruses, trichoviruses and foveaviruses belong to the family Betaflexiviridae.To empirically validate the new assay, grapevine material infected by ten different vitiviruses (i.e. GVA to GVL) was obtained from the Foundation Plant Services  pipeline of domestic and foreign introductions. A previous study in this grapevine collection revealed the presence of multiple vitiviruses . In addiFollowing the protocol described by , plant m2 (25 mM), 1 μl of dNTPs (10 mM of each), 1 μl each of Vitivirus-RdRp-F and Vitivirus-RdRp-R (50 μM), 0.25 μl of GoTaq G2 Flexi DNA Polymerase (5 U/μl)  and 12.75 μl of nuclease-free water. Amplification conditions consist of 3 min at 94°C, 35 cycles of 30 s at 94°C, 45 s at 53°C, 1 min at 72°C, and a final extension step of 7 min at 72°C. PCR products were electrophoresed on a 1.5% agarose gel in TAE buffer containing Gel Red nucleic acid gel stain .The universal assay consists of a two-step RT-PCR using the degenerate primers Vitivirus-RdRp-F and Vitivirus-RdRp-R. For reverse transcription (RT), a 20 ul reaction comprised of 7 μl of total RNA template, 4 μl of Invitrogen 5X First Strand buffer, 3 μl of random hexamer primers (0.1 μg/μl), 2 μl of dithiothreitol (0.1 M), 1 μl of dNTPs (10 mM each), 0.2 μl of RNasin Ribonuclease Inhibitor (40 U/μl) , 0.25 μl of SuperScript II Reverse Transcriptase (200 U/μl)  and 2.55 μl of nuclease-free water, was incubated at 25°C for 15 min, 42°C for 90 min and 72°C for 15 min. The PCR reaction includes 2 μl of RT product, 5 μl of Promega 5X GoTaq Green buffer, 2 μl of MgCl−5) of total RNA from two grapevines infected by GVA and GVB. The universal assay was compared against two commonly used RT-PCR assays specific for either GVA or GVB [Assay sensitivity was investigated using serial dilutions  following the manufacturer’s instructions. Ten clones per PCR transformation were Sanger sequenced employing M13 primers at the University of California-Davis DNA Sequencing Facility.Amplicons generated by the universal assay were purified using the DNA Clean & Concentrator -5 kit , cloned using the TOPO TA Cloning kit and transformed into One Shot Top 10 chemically competent cells of The Davis Virus Collection (DVC) located at the University of California-Davis contains 385 grapevines representing multiple domestic selections . Plants Vitivirus (GenBank taxid:184751), genus Trichovirus (GenBank taxid:40276) and genus Foveavirus (GenBank taxid:129725) using the BLASTp program [To determine if the developed RT-PCR assay can detect vitiviruses in other hosts, mint and blueberry material infected by mint virus 2 (MV-2)  and blue program .The search for regions of high homology in vitivirus nucleotide sequences and CP amino acid sequences were unsuccessful. MUSCLE alignment of REP proteins encoded by grapevine vitiviruses, including definitive and tentative species, did show a considerable level of conservation. Two conserved motifs (i.e. motif A and motif B) were identified in the RdRp domain which were long enough to generate PCR primers .Vitis sp. or GPGV- or GRSPaV-infected grapevines , MV-2 and BGMaV, revealed an 100% identity and coverage for five of these viruses; however, two vitiviruses, BGMaV and AVV had 100% identity and 87% coverage , including Vitivirus, Trichovirus and Foveavirus. After in-silico and in-vitro analyses of trichoviruses and foveaviruses, we did not find evidence for cross-reaction by the universal assay.Although y plants . For exaVitivirus species in grapevine and other hosts given its unbiased nature. Similar assays have been developed for carlaviruses [Betaflexiviridae [A single test for all known grapevine vitiviruses can be a useful tool for improving efficiency and reducing costs of large-scale surveys. Potentially, this generic assay may detect novel aviruses , nepoviraviruses  and diffiviridae .Grapevine is clonally propagated, consequently, to prevent the spread of vitiviruses, it is critical to use virus tested material. The assay developed here will be made available to diagnostic labs and will facilitate the production of certified virus-tested propagation material and the effective control of vitiviruses.S1 Table(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S1 Raw Images(PDF)Click here for additional data file.


Bungowannah virus is a pestivirus known to cause reproductive losses in pigs. The virus has not been found in other species, nor is it known if it has the capacity to cause disease in other animals. Eight sheep, eight calves and seven pregnant cows were experimentally infected with Bungowannah virus. It was found that sheep and calves could be infected. Furthermore, it was shown that the virus is able to cross the bovine placenta and cause infection of the foetus. These findings demonstrate the potential for species other than pigs to become infected with Bungowannah virus and the need to prevent them from becoming infected. Myocard species .Pestiviruses were initially classified according to their host specificity. Whilst this classification was originally appropriate for classical swine fever virus (CSFV), it was soon shown that bovine viral diarrhea virus (BVDV) and border disease virus (BDV) could naturally infect a variety of ruminants, pigs and other mammals. Recently, CSFV has also been shown to naturally infect cattle . In contA series of inoculation experiments were conducted in both sheep and cattle. Cattle were either directly inoculated using intranasal instillation or by co-housing with pigs that were chronically infected with Bungowannah virus. Sheep were either directly inoculated using intranasal instillation or subcutaneous injection or by co-housing with pigs that were chronically infected with Bungowannah virus. The specific details are as follows:The inoculum used for each of the direct inoculation experiments was derived from pooled pig foetal tissues that were passaged once in PK-15 cells . The titre of infectious virus was also determined by titration in PK-15 cells using standard methods.2HPO4, 2.7 mM KCl and 1.5 mM KH2PO4, pH 7.4) containing 0.5% gelatin (w/v), 5000 IU penicillin/mL, 95,000 IU streptomycin, 50 µg/mL amphotericin B and 0.1% (w/v) phenol red (PBGS). Swabs were collected into 3 mL of sterile phosphate buffered saline . The method has been previously described . The fluies/5 µL  was inclAntibody titres against Bungowannah virus were measured by virus neutralisation test (VNT). The VNT was performed as described previously . SelecteSheep used in these trials were obtained from a flock that was free of infection with ruminant pestiviruses and had not been vaccinated against pestiviruses. All sheep were tested for anti-pestivirus antibodies using a bovine viral diarrhea virus agarose gel immunodiffusion assay  and were10 TCID50/mL). Two other sheep were inoculated with the same dose subcutaneously while another two other sheep were held as uninfected controls. The inoculated sheep were held in two 11 m2 rooms . The two uninfected sheep were held in a similar 11 m2 room and were not challenged.Six 3-month-old Merino lambs were infected intranasally with 2 mL of cell culture amplified Bungowannah virus . Serum samples were tested for the presence of antibodies against Bungowannah virus using a VNT.2) with three pigs that were chronically infected with Bungowannah virus. The pigs  had been infected in utero and were shown to be shedding Bungowannah virus  7 days prior to the trial [Four 3-month-old Merino lambs were held in a small room . Two additional calves were held as uninfected controls and did not receive a challenge. The conditions under which they were held, the clinical examination and sampling were conducted as described above for the sheep.Eight 10-week-old Holstein–Friesian calves were infected intranasally with 2 mL of cell culture-amplified Bungowannah virus  [Four 5-week-old Holstein–Friesian calves were held with two pigs . Cows were between 53 and 65 days of pregnancy at the time of inoculation. Nasal and conjunctival swabs and serum samples were collected daily for 14 days following inoculation. The swabs and sera were tested for the presence of Bungowannah virus using qRT-PCR. Serum samples were then collected monthly until calving and were tested for antibodies against Bungowannah virus using a VNT. Pregnancy was monitored by ultrasound examination on a monthly basis. Cows were induced to calve between 255 and 276 days of pregnancy. Serum samples were collected from each calf after birth and prior to suckling. Conjunctival, nasal, oral and rectal swabs and serum samples were collected from calves every 48–72 h for 14 days. Vaginal swabs were also collected from the cows post-partum. A post-mortem examination of the calves was performed between 2 and 4 weeks of age. A wide range of tissues including brain, myocardium and skin  were tested for the presence of Bungowannah virus by qRT-PCR. Samples for qRT-PCR were collected by firmly rubbing a swab across the freshly cut surface of a section of the tissue and placed into 3 mL of PBGS. Skin biopsies were stored in 3 mL PBGS. All samples were stored at 4 °C until tested by qRT-PCR and virus isolation. Five pregnant Holstein-Friesian and two Illawarra-Shorthorn cows were chosen for the trial. They were obtained from a herd known to be free of infection with ruminant pestiviruses and had not been vaccinated against pestiviruses. All cows were tested for anti-pestivirus antibodies using a bovine viral diarrhea virus agarose gel immunodiffusion assay  and founThe trials described in this paper were approved by the Elizabeth Macarthur Agricultural Institute Animal Ethics Committee. The specific approvals were AEC M09/17 “Studies of the biology of Bungowannah virus infections (PMC) in sheep and cattle” (7 December 2009) and AEC M10/16 “Effects of Bungowannah virus infection in pregnant cattle” (22 December 2010).Bungowannah virus RNA was detected between days four and 11 in the nasal swabs of five of the six sheep exposed by intranasal instillation. Ct values ranged between 27.9 and 39.9 . Low levBungowannah viral RNA was not detected in oral, conjunctival or rectal swabs of any of the inoculated or control sheep, nor in serum or nasal swabs for the control sheep or those inoculated subcutaneously. None of the infected sheep developed clinical signs during the course of the infection. The rectal temperatures remained within normal limits. All eight sheep directly inoculated  developed Bungowannah virus-specific antibodies . Viral RNA was detected only in the nasal swabs of three of the four sheep during the period of co-mingling with the chronically infected pigs. The Ct values ranged from 36.1 to 36.4. Bungowannah virus RNA was not detected in serum or any other swabs during this time and was not detected in serum or any swabs after the pigs were removed from the room. None of the sheep developed Bungowannah virus-specific antibodies.Bungowannah virus RNA was detected in nasal swabs of all eight calves on at least three occasions between days two and 11 . Viral RViral RNA was detected in the nasal swabs of two of the four calves during the 48 h that they were housed in contact with chronically infected pigs. The virus was detected in the oral swab of another calf on day two and the nasal swab of this same calf on day eight. This calf was the only one of the four to develop antibodies against Bungowannah virus. Neutralising antibodies were first detected 16 days post-exposure. The antibody titre in this calf peaked at 2048 between 63 and 84 days post-inoculation. Bungowannah viral RNA was not detected in any other samples collected. Calves remained healthy throughout the study.Each of the seven cows inoculated with Bungowannah virus were found to shed Bungowannah virus RNA in nasal secretions. Shedding began in most cows at day three, with peak virus shedding between days seven and 10. One cow was still shedding viral RNA in nasal secretions on day 14 . BungowaAll cows developed neutralising antibodies against Bungowannah virus . One cowPregnancies in the cows were unremarkable with no abnormalities were detected by ultrasound. At birth and before suckling three of the seven calves were found to have antibodies against Bungowannah virus. Bungowannah virus RNA was also detected in ear skin biopsy samples from these same three calves, with Ct values of 26.0, 32.7 and 33.6. The calf with the highest RNA concentration  was the progeny of Cow 3. Bungowannah virus RNA was also detected in the serum of this calf. Viral RNA was not detected in conjunctival, oral, nasal or rectal swabs. All calves appeared normal at birth and displayed normal behaviour. The virus was detected in the vaginal swabs from dams of the three positive calves, but not from the other cows. Cow 3 had a Ct value of 30.4, while the other two were 35.8 and 36.6.Post-mortem examination of all calves was unremarkable, with no gross abnormalities detected. The three calves found to have Bungowannah virus RNA in the skin at birth were euthanased and necropsied at four weeks of age. Viral RNA was again detected in the skin of these calves, and also detected in the testicle of the single male calf among these three . Viral RIn this study we have demonstrated that Bungowannah virus can infect a proportion of both cattle and sheep after intranasal or subcutaneous inoculation, although infection was less efficient by the intra-nasal route when compared to pigs . AdditioNo significant disease was observed in the infected sheep and cattle, despite all the intranasally infected cattle shedding Bungowannah virus RNA in nasal swabs for up to two weeks after infection. The qRT-PCR results indicated that only low levels of virus were shed. The pattern of detection of RNA was consistent with virus replication rather than residual inoculum as there was a short period after inoculation when no RNA was detected. Similarly, viral RNA was detected in the nasal swabs of five of the six sheep infected intranasally, yet no disease was observed. It is interesting to note that the development of the humoral immune response in the two subcutaneously inoculated sheep occurred over a similar period as the intranasally inoculated sheep. We speculate the infection was localised at the injection site in these two animals and, in the absence of a viraemia, shedding via the upper respiratory tract did not occur. The low level of viral RNA detection in nasal secretions of sheep and cattle would suggest that ruminants are unlikely to play a significant role in transmission of the virus.It was also possible to infect a calf through co-habitation with a chronically infected pig for 48 h. Interestingly, this calf was the most curious of the four calves in the trial and interacted the most with the pigs. It is possible that if given longer exposure to the infected pigs the other calves may have become infected. Although all cows became infected, transplacental infection of the calf only occurred in less than half of the cases. Viraemia was associated with in utero infection in two of the four cows in which it was detected. It is noteworthy that Cow 3 developed a rising anti-Bungowannah virus antibody titre during the last trimester of pregnancy. The progeny of this cow was the calf with the highest levels of Bungowannah RNA at birth. We speculate that the dam’s immune system may have been stimulated by transplacental transfer of Bungowannah virus antigen during the last trimester of pregnancy.Each of the three calves that became infected had seroconverted by the time of birth. Persistent infections were not established, despite the cow being infected at a stage of gestation where immunotolerance and persistent infections would be expected, as usually occurs with BVDV infection. These three calves had significant levels of Bungowannah virus RNA present in skin samples at birth, over 210 days after their dams were infected. We hypothesise that these calves did develop a generalised infection in utero, but subsequently cleared the infection some time prior to birth. The skin and testes were the only sites where the viral RNA was not cleared. The testes are an immunoprivileged site, and so it may be that the infection continued at this site for a longer period of time, perhaps much closer to birth. It has also been suggested that skin is also immunoprivileged . It has The timing of infection of pregnant cows was designed to maximise the likelihood of producing persistently infected calves. Infection of cattle with BVDV during the first 90 days of gestation will generally produce calves that are immunotolerant to BVDV ,17. The Understanding why Bungowannah virus resulted in a humoral response in these calves may lead to a deeper understanding of the functional differences between specific proteins in BVDV and Bungowannah virus and how they do or do not affect evasion of the host innate immune response in the bovine or porcine foetus . In conclusion, while cattle and sheep can be infected with Bungowannah virus, they appear to be less susceptible to infection compared to pigs when challenged with a similar dose of virus. When compared to the infection of pigs , ruminan


Paramyxoviridae. Phylogenetic analysis groups Belerina virus together with the bat-borne Shaan virus within the genus Jeilongvirus, although expanding the tree with partial genomes shows Belerina virus forming a separate subclade within this genus, alongside a yet-unnamed paramyxovirus isolated from a greater tube-nosed bat. In summary, we discuss the complete genome sequence of Belerina virus, a putative new paramyxovirus species commonly found in Belgian hedgehogs.Common or European hedgehogs can be found throughout Western Europe. They are known carriers of a variety of parasitic and bacterial pathogens, and have also been shown to carry several viruses, including morbilli-like paramyxoviruses, although the pathogenic and zoonotic potential of some of these viruses has yet to be determined. We report here the discovery of a novel paramyxovirus in Belgian hedgehogs, named Belerina virus. The virus was detected by nanopore sequencing of RNA isolated from hedgehog tissue. Out of 147 animals screened in this study, 57 tested positive for Belerina virus (39%), indicating a high prevalence of this virus in the Belgian hedgehog population. Based on its divergence from other known paramyxovirus species, Belerina virus is thought to represent a new species in the family  Erinaceus europaeus), also known as common hedgehogs, can be found throughout Western Europe, stretching from southern Italy to southern Scandinavia and including the Iberian Peninsula1. They can live in a variety of habitats and are often found in gardens and parks. In recent years, hedgehogs have gained popularity as household pets, especially in the Western world, although this typically concerns four-toed hedgehogs , as the keeping of European hedgehogs is illegal in most Western countries4. Furthermore, wild hedgehogs that are underweight or have been injured are often housed by people aiming to help them regain their strengths and are sometimes kept as pets once they have recovered.European hedgehogs  and fungal  pathogens, and several cases of hedgehog-transferred zoonoses have been reported in the past decade9. Additionally, hedgehogs also harbor several zoonotic viruses, including rabies and tick-borne encephalitis virus, as well as other viruses of which the pathological and zoonotic potential remains to be fully elucidated13. An example of this is the finding of a paramyxovirus in European hedgehogs in 198114.Because of this close contact between humans and hedgehogs, some concerns have arisen in recent years about the potential of zoonotic diseases to be transferred from hedgehogs to humansParamyxoviridae is a family of single stranded, negative-sense RNA viruses that was recently divided into four subfamilies15. The largest of these subfamilies, the Orthoparamyxovirinae, is further subdivided into eight genera and comprises 34 recognized species16. In addition to fish and reptiles, members of this subfamily infect a wide variety of mammals17. Most orthoparamyxoviruses are known to cause disease in their respective hosts and some  are known zoonotic pathogens that can spread to humans19. Based on a neutralization assay using antisera against different paramyxoviruses, the aforementioned virus discovered by Vizoso and Thomas was thought to belong to the Morbillivirus group 14. The virus was isolated from the feces and lungs of a severely ill hedgehog and caused a symptomatology similar to Canine distemper virus, although it was also found in feces of multiple healthy animals. Unfortunately, the genome sequence of this virus was not determined.The family Erinaceus europaeus, which was detected in the context of a study aimed at virus discovery in different Belgian Eulipotyphla species. Belerina virus shows a relatively high prevalence in the hedgehogs screened in this study and appears to share similarities with paramyxoviruses found in bats.Here we report the complete genome sequence of Belerina virus, a putative new paramyxovirus species discovered in 20. This new virus was given the name ‘Belerina virus’, based on its location of origin (Belgium) and the identity of its host (Erinaceus Europaeus).We decided to use the Oxford Nanopore MinION to screen the total RNA, taken from the kidney of a European hedgehog, for the presence of previously undiscovered viruses. After running for 48 h, 5.1 M reads were produced, totaling 1.25 Gb after trimming. A tblastx search of all reads against the viral subset of the NCBI RefSeq database found 38 reads displaying limited similarity to different paramyxovirus genomes, indicating the presence of a novel paramyxovirus. Based on these 38 reads, we designed primer sets spanning the entirety of the viral genome and used these to determine the complete genome sequence of this virus, through a combination of PCR and 5′/3′ RACE, and subsequent Sanger sequencing. The complete genome (GenBank: MN561699) is 15,948 nucleotides long, thereby adhering to the so-called ‘rule-of-six’, which states that efficient replication of paramyxovirus genomes is dependent on their length being a multiple of sixJeilongvirus , groups Belerina virus within the genus rus Fig. a. This grus Fig. b. As a cParamyxoviridae typically share a similar genome organization, characterized by six major ORFs. These ORFs encode the nucleocapsid (N), a phosphoprotein (P), a matrix protein (M), two membrane glycoproteins (F and G) and the viral polymerase (L). Through a combination of leaky scanning (C) and mRNA editing , some additional accessory proteins are usually expressed from the P ORF. The V, W RNA editing site is marked by a motif sequence (YTAAAARRGGCA) that is conserved across all members of the genera Henipavirus, Morbillivirus, Jeilongvirus and Narmovirus, with the exception of Cedar virus and feline morbillivirus. Also in the genome of Belerina virus, this motif sequence is present (TTAAAAAAGGCA), indicating that mRNA editing likely also occurs in the case of Belerina virus, although this was not verified experimentally.Members of the family Jeilongvirus is that their genomes contain additional ORFs between the F and G ORFs. In the genomes of Mount Mabu lophuromys paramyxovirus 1/2 (MMLPV-1/-2), as well as in those of the yet-to-be-classified feline paramyxovirus and the unnamed bat paramyxovirus BtMl-ParaV (KJ641657), only one such ORF is present, encoding a transmembrane protein (‘TM’). In all other jeilongviruses, a second additional ORF is present, encoding a small hydrophobic protein (‘SH’). The role of these two proteins is not yet fully elucidated, although recent research has shown that for JV, the SH protein is involved in inhibiting the production of TNF-α, while the TM protein seems to stimulate cell-to-cell fusion23. A SH protein is also present in a limited number of other paramyxoviruses , while the TM protein is found exclusively in members of the genus Jeilongvirus. The genome of Belerina virus encodes only the TM protein, although this protein is slightly larger (310 aa) than the TM proteins (218–275 aa) of other jeilongviruses.A defining characteristic of members of the genus We collected kidney tissue samples from 147 Belgian hedgehogs that were brought in to three different animal rescue shelters. In total, 57 animals tested positive for Belerina virus (38.78%). A map showing the origin of the different animals is shown in Fig. The animals collected during the last year of sample collection (10) were selected for later histological analysis. A detailed overview of the histological findings is shown in Supplementary Table Jeilongvirus. Interestingly, Belerina virus does not encode an SH protein, like Shaan virus, but instead has the same unique genome structure as MMLPV-1/-2, containing seven major ORFs (N-P/V/C-M-F-TM-G-L). The phylogenetically basal position of these viruses, relative to other members of the genus Jeilongvirus, seems to suggest that jeilongviruses have acquired their additional ORFs sequentially, starting with the acquisition of the TM ORF, followed later by the addition of the SH ORF. Additionally, this SH-ORF acquisition appears to have occurred twice, once in the rodent-borne jeilongvirus lineage and once in the case of Shaan virus. When the phylogenetic analysis is expanded to also include likely paramyxovirus species for which only partial, yet largely complete (> 90%), genomes are known: rodent paramyxovirus, feline paramyxovirus and four additional  genomes of bat paramyxoviruses , Belerina virus now clusters with BtMl-ParaV, but separately from the other bat jeilongviruses. This clustering is also reflected in the genome organization of these viruses, with both Belerina virus and BtMl-ParaV lacking an SH ORF, while the four other bat virus genomes do presumably encode SH proteins, although both these SH proteins and these viruses’ TM proteins are significantly larger than the SH and TM proteins of other jeilongviruses . The paramyxovirus attachment glycoprotein, sometimes designated H(N) protein if the glycoprotein possesses hemagglutination (and neuraminidase) activity, is primarily involved in regulating the binding of virus particles to target cells14. Based on neutralization assays, they concluded that the virus they found was most similar to viruses of the Morbillivirus group . It is also stated briefly that similar viruses could be isolated from apparently healthy hedgehogs. Based on the information given in their report, it is possible that the virus they described belongs to the same species as Belerina virus, but this could not be verified due to a lack of sequence information.For this study, 147 animals from different locations throughout Belgium were screened for the presence of Belerina virus. Almost 40% of these animals tested positive. It is possible that this number is an overestimation of the actual prevalence due to sampling bias. The animals tested in this study were all brought into animal rescue shelters because they were wounded, weakened, sick or dead. If Belerina virus actually causes disease in hedgehogs, this method of sample collection could result in an overestimation of virus prevalence in the Belgian hedgehog population. However, histological analysis of two virus-positive animals revealed no virus-specific kidney or lung lesions, hinting at a limited or sporadic pathogenicity (if any) of Belerina virus. Unfortunately, we were unable to acquire additional tissue samples for more extensive histological analyses. Until now, there has been only one report of paramyxoviruses causing disease in hedgehogs. In their report, Vizoso and Thomas describe the finding of a paramyxovirus in a hedgehog displaying symptoms akin to canine distemperJeilongvirus.In summary, we report here the first description of Belerina virus, a novel paramyxovirus found in European hedgehogs. Belerina virus appears to have limited pathogenicity and seems to be prevalent in the Belgian hedgehog population. Comparison of the genome organization as well as phylogenetic analyses indicate that Belerina virus is most similar to a bat virus for which only a partial genome sequence is available, but that together these two viruses represent a separate clade within the genus A total of 147 hedgehog carcasses were collected from 2015–2019 from three animal shelters in Belgium . Further viral enrichment was achieved by layering 10 ml of filtered supernatant onto 2 ml of 30% (wt/vol) sucrose-PBS and centrifuging at 30,000 rpm for 3 h in an SW41 rotor 27. Following ultracentrifugation, total RNA was extracted using the QIAamp Viral RNA Mini kit . No carrier RNA was used. Any DNA present in the eluate was then removed enzymatically using TURBO DNase . Following a clean-up step using the RNeasy MinElute Cleanup kit (Qiagen), according to the manufacturer’s instructions, the purified RNA was amplified using the Complete Whole Transcriptome Amplification Kit WTA2 . The resulting cDNA was cleaned up using the MSB Spin PCRapace kit  and used to prepare a MinION sequencing library using the SQK-LSK108 kit , following the manufacturer’s recommendations. The sequencing library was ran for 48 h on a FLO-MIN106 flowcell (Oxford Nanopore Technologies). Adapter sequences were removed using Porechop (v0.2.4)28.Viral RNA was extracted from the kidney of one hedgehog by homogenizing 20 mg of renal tissue in 100 µl sterile PBS, using zirconium oxide beads, for two minutes at 4000 rpm using a Minilys homogenizer . The resulting homogenate was centrifuged at 17.000 Primers were designed, based on the sequences of the reads obtained by nanopore sequencing, to generate amplicons spanning the remaining gaps in the Belerina virus genome. Amplicons were generated using the OneStep RT-PCR kit (Qiagen). Used PCR conditions were: reverse transcription at 50 °C for 30 min, followed by 15 min at 95 °C and 40 cycles of 30 s at 94 °C, 30 s at 55 °C and 1 min at 72 °C, and a final extension step at 72 °C for 10 min. The resulting amplicons were purified using PureIT ExoZAP  and sent to Macrogen  for Sanger sequencing. Given the low single read accuracy of the nanopore data, the obtained Sanger sequences were used to design new primer sets, spanning the gaps between the previously obtained amplicon sequences, allowing for the resequencing of all used nanopore data. PCR cycling and Sanger sequencing was done as described above. All used primers are listed in Supplementary Table Determining the exact 5′ and 3′ ends of the Belerina virus genome was done using the 5′/3′ RACE kit 2nd generation , replacing the standard Oligo dT-Anchor Primer by a slightly modified one that also targets the conserved trinucleotide found at the 3′/5′ ends of paramyxovirus genomes (GAC CAC GCG TAT CGA TGT CGA CTT TTT TTT TTT TTT ACC). For the 3′ end, a poly-A tail was added to the virus genome prior to cDNA generation, using the Poly(A) Polymerase Tailing Kit  according to the manufacturer’s instructions. For both ends, amplification of the poly-A tailed RNA/cDNA was done with the OneStep RT-PCR kit (Qiagen). See Supplementary Table For all 147 hedgehogs, viral RNA was extracted from one of the kidneys using the RNeasy Mini kit (Qiagen), according to the manufacturer’s protocol. All extracts were screened for the presence of Belerina virus using a specific primer set targeting a 214 bp region in the Belerina virus L gene  with the gappyout setting30. Following selection of an LG + G + I model, using ProtTest (v3.4.2), as an adequate model to describe the amino acid substitution process, BEAST v1.10.4 was used to infer Bayesian phylogenetic trees33. Markov chain Monte Carlo analyses were ran until adequate effective sample sizes (ESS > 200) were obtained. TreeAnnotator was used to summarize a maximum clade credibility tree from the posterior tree distribution, employing a burn-in of 10%. FigTree v1.4.3 was used to visualize the resulting tree.The complete amino acid sequences of the N, P, M, F, G and L open reading frames (ORF) of all publicly available complete and near-complete orthoparamyxovirus genomes were aligned separately using MAFFT (v7.123b) and subsequently concatenatedSupplementary Information.


Escherichia coli biofilm matrix protein CsgA was initially genetically fused with the influenza‐virus‐binding peptide (C5). The resultant engineered living biofilms could correspondingly capture virus particles directly from aqueous solutions, disinfecting samples to a level below the limit‐of‐detection for a qPCR‐based detection assay. By exploiting the surface‐adherence properties of biofilms, it is further shown that polypropylene filler materials colonized by the CsgA‐C5 biofilms can be utilized to disinfect river water samples with influenza titers as high as 1 × 107 PFU L−1. Additionally, a suicide gene circuit is designed and applied in the engineered strain that strictly limits the growth of bacterial, therefore providing a viable route to reduce potential risks confronted with the use of genetically modified organisms. The study thus illustrates that engineered biofilms can be harvested for the disinfection of pathogens from environmental water samples in a controlled manner and highlights the unique biology‐only properties of living substances for material applications.Waterborne viruses frequently cause disease outbreaks and existing strategies to remove such viral pathogens often involve harsh or energy‐consuming water treatment processes. Here, a simple, efficient, and environmentally friendly approach is reported to achieve highly selective disinfection of specific viruses with living engineered biofilm materials. As a proof‐of‐concept,  A simple, efficient, and environmentally friendly approach to achieve highly selective disinfection of specific viruses with living engineered biofilm materials from virus‐polluted water is reported. The engineered biofilms can disinfect influenza virus from contaminated water to a level below the limit‐of‐detection for a qPCR‐based detection assay, thus protecting mammalian cells against virus infections efficiently. Conventional technologies to address this problem like chlorine and ozone treatment are chemically intensive and may produce dangerous disinfection byproducts, while use of UV light illumination and high‐pressure filtration are energy intensive and can fail against UV‐resistant viruses like adenovirus. Further, some viruses are resistant to the chemicals used in water disinfections, and the sizes of some viruses are too small to be filtered by conventional membranes. Thus, the development of a new generation of simple, efficient and environmentally friendly virus disinfection strategies that are complementary to existing technologies would be highly demanded. To this end, water treatment experts have suggested the exciting possibility of future technologies that might achieve exquisite molecular‐level specificity for selective viral binding to materials functionalized with, for example, host receptor proteins of specific viruses.Waterborne disease outbreaks from viral pathogens occur each year worldwide,1 and —have been used in water treatment for a long time. For example, naturally occurring biofilms are frequently harvested for the remediation of toxic compounds and heavy metals. Inspired by these historical applications of biofilms in water treatment, we here propose and explore the concept of engineering biofilms as living materials for virus disinfection based on the extracellular assembly of genetically engineered proteins in the biofilm matrix to enable specific interactions with and thus robust capture of targeted pathogenic viruses. Ideally, such an approach would achieve highly efficient and selective disinfection of targeted viruses with very low energy inputs and minimal infrastructure requirements. Moreover, this living biofilm platform would harness the unique properties of living systems, including genetic programmability, self‐regeneration, and evolutionary and environmental adaptability, attributes offering advantages over conventional water treatment technologies in terms of scalability for biomanufacture and deployment, for example, to prevent transmission of waterborne viral pathogens at geographically remote or otherwise inaccessible sites during epidemic outbreaks.Biofilms—consortia of microbial cells embedded in a protective extracellular matrix9—have2Escherichia coli biofilms for disinfection of virus in water was based on CsgA proteins, a major component of E. coli biofilms. CsgA protein monomers are secreted from bacterial cells and can self‐assemble into amyloid nanofibers. Notably, genetically modified bacterial biofilms have recently found a wide range of interesting applications in catalysis, biosensor, and bioremediation as engineered living materials. As an initial proof‐of‐concept for viral binding in this study, we choose the influenza virus (H1N1) as a model, and engineered fusion monomers that combined CsgA with a known influenza‐virus‐binding peptide—here denoted as C5—which was previously identified via phage display; C5 (amino acid sequence: ARLPR) has been shown to bind to hemagglutinin (HA), a membrane glycoprotein of influenza virus . Molecular dynamics simulations of the interaction between a CsgA‐C5 fusion monomer and hemagglutinin by GROMACS indicated that these two proteins interact strongly: the bound complex structure was stable even after 800 ns  and western blotting . We then conducted quartz crystal microbalance (QCM) experiments wherein fresh eluted CsgA and fusion CsgA‐C5 monomers were exposed to silicon substrates that were coated with hemagglutinin. Compared with CsgA control monomers, CsgA‐C5 monomers showed substantially enhanced absorption: the mass of CsgA‐C5 monomers absorbed on the HA‐coated substrate was about four times as much as that of the absorbed CsgA monomers   and Congo red assays  showed that CsgA‐C5 and CsgA proteins exhibited amyloid features. Further, both the CsgA‐C5 and CsgA monomers were able to self‐assemble into long and stable fibers, as confirmed by transmission electron microscopy (TEM) and atomic force microscopy (AFM)  , however, the affinity of CsgA‐C5 fibers for hemagglutinin was increased by tenfold compared to CsgA fibers  to test if the self‐assembled amyloid nanofibers retained their capacity for binding hemagglutinin. We first quantified the biomass densities of the CsgA and CsgA‐C5 nanofiber samples formed on the substrates by measuring the initial and remaining concentrations of protein solutions before and after nanofiber coating formation on the substrates. Note that the biomass densities of the CsgA and CsgA‐C5 nanofiber coatings were identical . We collected the supernatants from the samples, and AFM analysis showed that there were many virus particles in the control supernatants (from CsgA biofilm samples) but hardly any in the supernatant from the CsgA‐C5 biofilm samples, even at very high viral titers . Further, ELISA detected a clear concentration‐dependent increase in viral signal for the control samples that were not exposed to biofilms (3 days at 29 °C), while only a very low signal was detected for the supernatants from the CsgA‐C5 biofilm samples, with a slight increase evident for only the highest titer sample  cells to control or post‐biofilm incubation supernatant from the 7 × 10n Figure . A similE. coli biofilms can inherently adhere to diverse substances and complex structures, we next grew CsgA‐C5 biofilms on the polypropylene surfaces of intricate gear‐like industrial filler material and tested their capacity to capture virus particles from river water . We next spiked river water samples with an influenza virus titer of 1 × 107 PFU L−1, a level much higher than reported human virus titers in river water (which range from 102 to 105 PFU L−1). We then passed the water samples over the filler materials, and qPCR analysis showed that the virus could be easily detected in the unfiltered control samples (virus‐spiked river water) but was undetectable in the filtrate sample  at room temperature for at least 2 h. After sufficient binding between CsgA‐C5 biofilms and virus particles, the solution in the column was allowed to flow out at an average rate of 0.67 mL s−1. The titers of virus solution were next determined by qPCR assay, and the results showed that the titers changed from 1.84 × 105 to 7.3 × 104 PFU mL−1 after the virus solution was treated with CsgA‐C5 biofilm‐coated fillers. The total capacity of the fillers could be calculated with a value of 1.11 × 106 PFU. Therefore, the maximum capacity of the filtration unit could be determined with an average value of 2.22 × 105 PFU per filler. We next used five similar pieces of biofilm‐coated fillers to treat virus solution at a titer of 1.0 × 104 PFU mL−1 for assessing the amount of samples that one filtration unit process before its virus capture capability drops. Our results indicated that this system could at most treat a total of 50 mL influenza virus‐polluted water (10 mL per filler): when the volume of virus solution applied increased above 50 mL, the capacity of virus disinfection from river water using the biofilm‐coated fillers dropped, as revealed by qPCR assay (cycles < 35) .To determine the maximum capacity of the filtration unit, we incubated five pieces of the CsgA‐C5 biofilm‐coated industrial fillers (diameter = 11 mm) in the presence of 10 mL excessive amount of influenza virus particles in river water , activated by the expression of LuxI‐LuxR, was introduced into our engineered E. coli. The luxI gene under a J23108 constitutive promoter, producing an acylhomoserine lactone , can activate the suicide gene expression regulated by LuxR repressor. In this way, when the cell density reaches a critical threshold, expression of the toxin protein (ϕx174E) will be initiated, thus resulting in bacterial death. As is shown in figure S14 in the Supporting Information, the growth curve of the engineered bacteria containing ϕx174E clearly showed an autonomous inhibition effect when cell density (OD600) reached 0.35, while the growth of the control bacteria (without a suicide gene circuit) showed a continuous growth mode without any inhibition effect . To test whether introduction of the suicide gene circuit would affect the performance of virus capture, we incubated the engineered bacteria containing the suicide gene with virus particles (3.5 × 107 PFU L−1) and found that the engineered bacteria could still efficiently express biofilms and effectively eliminate the virus particles in the supernatant . Collectively, these results thus showed the feasibility of introducing a suicide gene circuit for controlling the fate of engineered bacterial, thus illustrating a viable route to address the biosafety concerns raised by the use of genetically modified organisms including engineered bacteria.After confirming the feasibility of water disinfection using living engineered biofilms, we turned to assess the potential biosafety concerns brought about with the use of genetically modified organisms (GMOs). To such end, we engineered a quorum sensing‐enabled suicide gene circuit into our engineered bacteria.33 Spe Alternatively, another possible approach is to genetically delete the cell‐wall synthesis gene in the engineered strain , rendering the growth of the engineered bacterial dependent on exogenous diaminopimelate (DAP). In this way, any escaped engineered cells would not survive in the environment without providing sufficient DAP, as DAP in the surroundings is insufficient to support the growth of auxotrophs.We here used biofilms programmed with a virus‐protein‐binding peptide to endow engineered biofilms the ability to efficiently capture viruses from river water. Compared with conventional water treatment methods, our strategy is green, low costs, and low energy inputs. Essentially, our engineered CsgA‐C5 biofilms achieved strong and specific disinfection of the target virus from water samples to a level that precluded infection of cells known to be highly susceptible to influenza infection. Extending the concept to showcase the biology‐specific functional properties of a genuinely living material, we used our functional biofilms to grow on and colonize polypropylene inserts in a way that also robustly disinfected viruses from river water samples. Our initial proof‐of‐concept demonstrations targeted the influenza virus as a model, but clearly any virus‐binding peptide or protein‐based moiety  should be suitable for fusion with CsgA proteins to enable similar biofilm‐mediated disinfection for other viruses. To reduce potential risks confronted with the use of genetically modified organisms, we have applied a suicide gene circuit in our engineered strain that can successfully restrict the growth of bacterial in a controlled manner. Beyond strictly controlling bacterial growth, other gene circuits such as “deadman” and “passcode” could also be possibly engineered into the bacteria to control cell viability.34 AltVibrio cholera and protozoans like Giardia lamblia). It should also be possible to further engineer the material performance of the biofilms themselves by fusing the CsgA‐pathogen binding monomers to other protein domains that can alter biofilm mechanical properties tailored for specific applications. Looking beyond E. coli and in light of our previously reported work demonstrating that the FDA generally‐regarded‐as‐safe bacterium Bacillus subtilis and its TasA amyloid proteins can be engineered and generally functionalized as a biofilm living material platform, we anticipate that this generally pathogen‐binding biofilm concept could even find applications in other application domains, for example, applying engineered biofilms in the gut to capture and digest toxic gut pathogens or viruses.Given that bacterial biofilms can be genetically modified and considering the modularity of fusion amyloid monomers, it should be relatively straightforward to deploy other CsgA fusion proteins which combine the amyloid nanofiber self‐assembly and biofilm‐forming capacities of CsgA domains with additional functional domains that selectively bind to (and thus sequester) other viruses and perhaps even other classes of waterborne pathogens , especially in difficult‐to‐access areas during epidemic outbreaks. That is, rather than requiring the transport of dangerous chemicals, energy‐intensive filtration equipment, strong generators, and trained personal to properly and safely implement and manage pathogen‐disinfection water treatment processes, local inhabitants of such areas could for example grow living engineered biofilms in their own buckets and other water vessels.The authors declare no conflict of interest.J.P. and Y.L. contributed equally to this work. C.Z. directed the project. J.P. conceived the technical details and designed the experiments. J.P. carried out the experiments including plasmid construction, protein purification, TEM, AFM, ELISA, immunofluorescence, biofilm culturing, and data analysis. Y.L. performed the experiments including RNA extraction, qPCR, cell culture and virus infection, immunofluorescence, molecular dynamics, and data analysis. S.Z. and K.D. helped the molecular dynamics and related computational biology analysis. Z.C. and C.Q. helped the experiment part of cell culture and virus infection, B.A. constructed the plasmids. J.P., Y.L., and C.Z. analyzed the data, discussed results, and wrote the manuscript with help from all the authors. All the authors revised the manuscript.Supporting InformationClick here for additional data file.


Cryptococcus neoformans VNI and 16 C. gattii VGII isolates, revealing an endemic pattern in domestic microenvironments. The International Society for Human and Animal Mycology (ISHAM) consensus multilocus sequence typing (MLST) scheme for the C. neoformans/C. gattii species complexes identified two sequence types (STs), ST93 and ST5, amongst C. neoformans isolates and six STs amongst C. gattii isolates, including the Vancouver Island Outbreak ST7 (VGIIa) and ST20 (VGIIb), the Australian ST5, and ST264, ST268 and ST445, being unique to the studied region. Virulence studies in the Galleria mellonella model showed that five C.gattii strains and one C. neoformans strain showed a similar pathogenic potential to the highly virulent Vancouver Island outbreak strain CDR265 (VGIIa). The findings of this study indicate that humans can be exposed to the agents of cryptococcosis via house dust, forming the basis for future studies to analyze the impact of early and continuous exposure to indoor dust on the development of subclinical or clinical infections.Cryptococcosis, a potentially fatal mycosis in humans, is acquired via exposure to exogenous environmental sources. This study aimed to investigate the frequency, genetic diversity, and virulence of cryptococcal strains isolated from indoor dust in the Rio Negro micro-region of the Brazilian Amazon. A total of 8.9% of the studied houses were positive, recovering nine  Cryptococcus, the Cryptococcus neoformans species complex and the C. gattii species complex [C. neoformans is cosmopolitan, affecting mainly immunocompromised individuals, especially HIV-infected patients, with an estimation of 223,000 new cases of cryptococcal meningitis alone each year in this group of patients [C. gattii, which was previously associated with tropical and subtropical climates, predominantly causes a primary infection in immunocompetent individuals [C. gattii shows a regional pattern in Brazil, being mostly reported in the north and northeast of the country, where it occurs usually in immunocompetent hosts, including children and young adults [C. gattii appeared in British Columbia, Canada; at first, cases appeared in the form of outbreak related strains, later becoming endemic and persisting until today. Until 2015, 393 cases of cryptococcosis were reported in Canada, and currently Vancouver Island has one of the highest annual incidences of C. gattii infections amongst humans and animals in the world, making this region an important temperate endemic area of cryptococcosis [Cryptococcosis is a potentially fatal respiratory and neurological mycosis affecting humans and animals worldwide. The disease is caused by two pathogenic members of the genus  complex . Cryptocg adults . In 1999C. neoformans can readily be isolated from pigeon guano and has been shown to grow and mate on mediums containing this substrate [C. gattii has not yet been isolated from pigeon excreta, but as has C. neoformans, it has instead been recovered from various tree species [Cryptococcosis is acquired by the inhalation of infectious propagules (desiccated yeasts cells or basidiospores) from the environment . As suchubstrate , C. gatt species .C. gattii in dwellings in the hinterland of the Brazilian Amazon identified the genotypes ST7/VGIIb and ST20/VGIIa [Pioneering studies on cryptococcosis in AIDS patients in Central Africa and Brazil demonstrated the risk of these patients acquiring cryptococcosis from indoor dust ,11. In B20/VGIIa , which h20/VGIIa . However20/VGIIa .The present study in the Rio Negro micro-region, which is composed of four municipalities in the Amazon in Brazil, aimed to analyze the frequency, genetic diversity and virulence traits of the agents of cryptococcosis isolated from indoor dust.The study was conducted in the Amazonas state A in the C. neoformans and C. gattii. After sweeping the house with a broom from each residence, one indoor dust sample per household was obtained. The study had the approval of each city’s Health Department, as well as ethical approval by the National Institute of Infectious Diseases Ethical Research Committee, Rio de Janeiro, Brazil (Reference No. CAAE 23238913.3.0000.5262). Seventy-nine samples of indoor dust were obtained from the three cities of the Rio Negro micro-region: 51 from Santa Isabel do Rio Negro, 12 from Barcelos and 16 from Novo Airão. Cryptococcal isolates were recovered as described previously [Indoor dust was collected from houses in the three municipalities in different neighborhoods of each city to investigate the presence of eviously ,13. BrieC. neoformans and C. gattii were distinguished on canavanine-glycine-bromothymol blue (CGB) medium [Brown colonies were recovered and tested for urease production on Christensen urea agar and for carbon and nitrogen compound assimilation using VITEK 2-BioMerieux System . The species ) medium .C. neoformans/C. gattii species complexes, including the following seven unlinked genetic loc: CAP59, GPD1, LAC1, PLB1, SOD1, URA5 and the IGS1 region [After DNA extraction , the mat1 region . The seq1 region . All newThe genetic relationships of the 7 concatenate MLST loci were shown using the software Splitstree4 v. 4.14.5 . UnrooteG. mellonella were used to evaluate the virulence of selected cryptococcal isolates recovered in this study. Larvae were obtained after the oviposition of adult moths reared and preserved at 26 °C and 60% relative humidity in the insectarium of the Westmead Hospital Animal Care Facility, Sydney, Australia. Ten larvae of similar size (about 3 g each) were selected, placed in a 90 mm plastic Petri dish and weighed before inoculation. Each fungal strain was grown on Sabouraud agar for 48 h at 27 °C. After cell counting using a Neubauer Chamber, an inoculum of 108 yeast cells/mL was prepared in phosphate buffered saline (PBS), from which 10 µL were inoculated into the hemocoel of each larva by injection into the last left pro-leg, using a 50U insulin syringe with a 29-gauge needle. To monitor potential effects on survival due to physical injury, a group of 10 larvae was also inoculated with PBS, while another 10 larvae were not inoculated as a non-infected control. One group of larvae, inoculated with the well-characterized highly virulent strain C. gattii CDCR265, was included as a reference to determine the degree of virulence of the isolates. After injection, the larvae were incubated in Petri dishes at 37 °C for 10 days and checked daily for any mortality [Larvae of the moth ortality .Survival curves were graphed, median survival times were calculated and the estimation of differences in survival was analyzed by the Log-rank (Mantel-Cox) test (CI 95%) per strain. Median survival times were not determined (ND) when more than five larvae (50%) were alive at the end of the experiment. Statistical analysis was performed using GraphPad Prism version 7.00 for Windows .From the 79 indoor dust samples taken, seven (8.9%) were positive for the agents of cryptococcosis: 3 from Santa Isabel do Rio Negro, 2 from Barcelos and 2 from Novo Airão .C. neoformans VNI and 16 C. gattii VGII isolates were recovered and genotyped using the ISHAM consensus MLST scheme for the C. neoformans/C. gattii species complexes [C. neoformans VNI isolates, ST93 (6/9) and ST5 (3/9) were identified, showing a clonal population. These two STs have previously been recovered worldwide. The most common C. gattii sequence type was ST7 (5 out of 16) (Vancouver Island Outbreak subtype VGIIb) and ST20 (2 out of 16) (Vancouver Island Outbreak subtype VGIIa). In addition, ST5 (1/16); previously described from Australia, and ST264 (2/16); ST265 (1/16); ST266 (1/16); ST267 (1/16); ST268 (1/16) and ST445 (2/16), unique STs to this region, were also identified.From the seven positive samples, nine C. gattii VGII population, which identifies the genetic diversity in this Amazon region, is shown in MATα. Only one MATa isolate amongst the C. gattii isolates was found, strain SI443-17 (ST268).The analysis of the combined MLST loci showing the placement of the 16 isolated strains from the Rio Negro micro-region in the context of the STs obtained from the global G. mellonella larvae infected with C. neoformans VNI environmental isolates was slightly higher compared to that of the larvae infected with the C. gattii VGII environmental isolates, although without a statistical difference between the survival curves of the infected larvae . Amongst the VGII isolates, however, five isolates were of comparable virulence  to that of the Vancouver Island outbreak strain CDCR265. One isolate of ST7 (VGIIb), both isolates of ST20 (VGIIa) and the isolates with ST266 and ST267 demonstrated high virulence in vivo  belonging to the ST93 were as virulent as the VGIIa highly virulent Vancouver Island outbreak strain CDCR265 (p > 0.05)  .Cryptococcal infection is acquired through human and animal exposure to exogenous sources, as such the understanding of the dynamics and adaptation of these environmental reservoirs is of fundamental interest to seek answers to two main questions: 1. How do humans get infected? and 2. What are the means to avoid or reduce risks of infection? Humans are primarily exposed to organic indoor air inhalation, and as such, indoor dust is an important mechanism of exposure, as people spend over 86.9% of their lives in indoor environments . StudiesCryptococcus species/strains are scarce. Pioneering studies in Central Africa in the late 1980s detected a large number of C. neoformans-positive indoor dust samples in households of patients with AIDS-associated cryptococcosis [C. neoformans. In addition, the authors observed that cryptococcosis was twice more frequent among AIDS patients residing in positive dwellings, thus suggesting an important role of positive indoor dust samples in the acquisition of cryptococcal infection in HIV/AIDS patients [Studies on emerging contaminants in indoor dust and the resulting levels of human exposure to pathogenic coccosis ,25. Anotpatients .C. gattii isolates in indoor dust associated with wooden houses [C. gattii acquired from the domestic environment. In the present study, a larger area including two more adjacent cities (Barcelos and Novo Airão) in the same Amazon region  revealed n houses , suggestn region  showed tC. gattii manifesting as CNS infections in immunocompetent young adults and children of both sexes in the Amazon and the northeast regions of Brazil is common, with the associated lethality ranging from 35% to 40% [C. gattii in indoor dust in the Rio Negro micro-region could explain the endemic pattern of cryptococcosis by VGII in children and young adults and thus indicates the need for future studies in other endemic areas to understand the role of the agents of cryptococcosis in indoor environments.An indoor microbial study revealed special concerns for vulnerable groups, such as children, for the risk of indoor-acquired infection . In Braz% to 40% ,28,29,30C. gattii VGII Brazilian population previously described and strongly supports the emergence of virulent strains from ancestors in the northern region of Brazil [The molecular subtypes of cryptococcosis responsible for the epidemic in Canada were first described in 1999 , but they have since become endemic in the region. In subsequent studies, the same subtypes were identified in the Amazon region in different cities and at different time points . ST7  and one genotype of C. neoformans VNI (ST93) presented a similar pathogenic potential to that of the highly virulent Vancouver Island outbreak strain CDR265  [During the present study, there was no evidence of cryptococcosis cases in the inhabitants of the positive dwellings. However, the Ca, ST20) .Cryptococcus, being the third most abundant genus in such samples [Cryptococcus spp. are non-pathogenic to humans. The present study showed that pathogenic Cryptococcus strains can also be found in indoor dust, posing a risk of cryptococcal infection for the inhabitants of the dwellings. The herein obtained findings reinforce the interplay of pathogenic fungal disease agents in the indoor environment as a possible source of infections of humans and animals, showing the importance of the application of the one health concept as an approach to understanding and counteracting fungal infections, especially in the immunocompromised  or other vulnerable populations (children and the elderly), by assessing the indoor environmental health situation of those individuals to prevent potentially life-threatening fungal infections.Microbiome studies of indoor dust and outdoor air samples in Boston and California (USA) detected a moderate percentage of the genus  samples ,35. ThesIn summary, the current study specifically points out the possibility of an exposure of humans to the causative agents of cryptococcosis in house dust in the Rio Negro micro-region of the Brazilian Amazon, where the main genotypes described worldwide are present and show a high virulence. Our findings also point towards the need for a thorough investigation of the importance of the presence of highly virulent disease agents in indoor dust microenvironments. Future studies are now warranted to analyze the impact of early and continuous exposure to indoor dust on the development of subclinical or clinical cryptococcal infections amongst the inhabitants of those houses and to illustrate the impact of interaction between humans, animals and the environment as part of the one health concept and the implications of this for the development of cryptococcosis.


Infection of bulls with bovine viral diarrhoea virus (BVDV) can result in the development of virus persistence, confined to the reproductive tract. These bulls develop a normal immune response with high neutralizing antibody titres. However, BVDV can be excreted in the semen for a prolonged period. Although relatively rare, in this study we describe six separate cases in bulls being prepared for admission to artificial breeding centres. Semen samples were tested in a pan-Pestivirus-reactive real-time PCR assay and viral RNA was detected in semen from five of the bulls for three to eight months after infection. In one bull, virus was detected at low levels for more than five years. This bull was found to have one small testis. When slaughtered, virus was only detected in the abnormal testis. The low levels of BVDV in the semen of these bulls were only intermittently detected by virus isolation in cell culture. This virus-contaminated semen presents a biosecurity risk and confirms the need to screen all batches of semen from bulls that have been previously infected with BVDV. The use of real-time PCR is recommended as the preferred laboratory assay for this purpose. Pestivirus A, and BVDV type 2 (BVDV-2) has been classified as Pestivirus B. These viruses are predominantly pathogens of cattle, though sheep, other ruminants and pigs may also become infected.Pestiviruses are a group of viruses that belong to the family Flaviviridae and are of animal health and economic importance worldwide . The taxTransmission of BVDV occurs horizontally by direct and indirect contact between cattle, and vertically by transplacental infection of the foetus. The virus replicates in mucosal epithelium and associated lymphoid tissues. A viraemia follows with subsequent viral replication throughout the body. Post-natal transient infections are often subclinical, but can result in a range of clinical conditions including reproductive losses, immune suppression, respiratory disease and acute diarrhoea . Pericon4 to 107.6 TCID50/mL) [The detection of BVDV in the semen of bulls is well documented ,7,8,9,10ID50/mL) . Semen wID50/mL) ,16.0.9 to 101.8 TCID50/mL) but can occasionally infect a susceptible female [Secondly, post-pubertal bulls exposed to BVDV undergo a transient infection and may shed virus in semen for up to 14 days , or perh3.3 TCID50/mL) for the rest of his life.Thirdly, some post-pubertal bulls exposed to BVDV become acutely infected but develop a prolonged or perhaps persistent testicular infection. These bulls are not PI, become transiently infected and mount a normal antibody response. The virus is cleared systemically, yet the virus is excreted in semen for prolonged periods. For this study, we have defined this prolonged testicular infection as the detectable presence of BVDV RNA in semen more than 60 days from when the systemic infection occurred. Until recently, these prolonged testicular infections (PTI) were considered to be rare. The first case was reported in 1998 . This buPTIs were induced experimentally in 2003 by inoculating susceptible post-pubertal bulls with BVDV . FollowiThe incidence of PTI is considered to be very low and few have been described ,18,19, yBlood samples and commercially prepared, extended semen from six bulls were submitted to the Virology Laboratory at the Elizabeth Macarthur Agriculture Institute (EMAI), New South Wales, Australia by veterinarians from artificial breeding centres. These samples were tested to meet regulatory protocols for the export of semen, where the serological status of an animal is determined and, if seropositive, their semen was subjected to virus detection. Subsequent to testing of the initial samples from these bulls, both archival samples and prospective collections were requested.Bull 2 was sent to an abattoir where the testes were collected and sent to EMAI. The testes were measured, weighed and dissected. Cut surfaces from various positions within the testes were swabbed using cotton tipped swabs which were placed into 3 mL of phosphate buffered gelatin saline (PBGS) and tested with a pan-Pestivirus-reactive qRT-PCR.A virus neutralisation test ,22 basedSerum from each of the bulls was tested to determine if the bull was persistently infected with BVDV using a commercial antigen capture ELISA  run according to the manufacturer’s instructions.The detection of BVDV RNA was performed by qRT-PCR as described previously . BrieflySemen samples were processed  and viruSections (4 µm thickness) of formalin fixed testes from Bull 2 were cut, mounted on Superfrost Plus slides  and oven-dried for 30 min. Sections were de-waxed in xylene and rehydrated in an ethanol series. Endogenous peroxides were then blocked with a 1.8% solution of peroxide in methanol for 20 min. Slides were washed briefly in distilled water followed by 2 × 5 min washes in phosphate buffered saline (PBS) with 0.05% Tween 20 . Antigens were unmasked at 37 °C for 15 min in undiluted Proteinase K . Sections were washed twice with distilled water and once with PBS/0.05% Tween 20 before being blocked for 1 h with 0.5% bovine serum albumin  and 1% normal goat serum in PBS. Following a rinse in PBS/0.05% Tween 20, 150 µL of mouse MAb 15C5  at a 1/10 dilution was added to each section and incubated at room temperature  for 1 h. The sections were washed twice in PBS/0.05% Tween 20 and incubated in EnVision™ anti-mouse serum  at RT for 1 h. Following three PBS/0.05% Tween 20 washes, 200 µL Dakocytomation DAB+  chromogenic solution was added to each section and developed for 10 min in the dark. Slides were rinsed with distilled water to stop the colour reaction, counterstained with Mayers haematoxylin, dehydrated and mounted with dibutylphthalate polystyrene xylene (DPX) mounting medium .Bos taurus breeds (95.9%), with Bos taurus × Bos indicus breeds (2.5%) and Bos indicus breeds (1.6%) making up the remainder. Neutralizing antibodies to BVDV were detected in 295 (50.3%) of these bulls at the time of screening. Semen samples from all bulls that had virus neutralizing antibodies to BVDV were tested by qRT-PCR.Bulls that enter semen collection centres are shown to not be viraemic for BVDV and neutralizing antibody titres are determined. A review of records held at EMAI indicated that during the period November 2012 to October 2019, 586 bulls were tested at EMAI for entry to artificial breeding centres. The majority of bulls in this group were Six bulls were identified to have PTIs because BVDV RNA was detected in semen more than 60 days after the first detection of infection. The 60-day limit provided sufficient time to elapse to exclude the possibility of residual RNA or virus from a recent acute infection being detected. All of these bulls gave negative results in the antigen ELISA, confirming that none was persistently infected. All bulls had high neutralizing antibody titres during the observation period. Live attenuated Pestivirus vaccines are not used in Australia and none of the bulls in this study had been vaccinated. Full details for the six infected bulls are described in Table A6Both beef (five) and dairy (one) bulls were involved, with ages at the time of first detection of virus infection ranging from 10 to 21 months . BVDV RNThe data for Bull 2  providesEach of these bulls with a presumed PTI was clinically normal and without any physical abnormalities, with the exception of Bull 2. Soon after arriving at the artificial breeding centre, this bull had scrotal measurements recorded on two occasions, with a circumference of 31 cm on 20 February 2012 and 34 cm on 16 April 2012. These measurements were considered normal for a bull of his breed and age . Six yeaWhen examined microscopically, the abnormal testis from Bull 2 showed multifocal but locally extensive areas of seminiferous tubular atrophy A,C charaInternational semen collection protocols and the associated health certification of donor bulls  are desiStudies of transiently infected bulls in the past have relied on virus isolation for the detection of BVDV in the semen. The use of qRT-PCR to detect BVDV in semen is recommended by the OIE , yet theIn 1998, Voges et al. described a post-pubertal bull that was shown to shed BVDV in semen over an 11-month period. This bull had a consistently high antibody titre against BVDV and yet was not viraemic and virus could not be detected in any organ apart from the testes. This bull was described as being in good health until his slaughter at 22 months of age. His growth rate and testicular development were unremarkable. Two other cases of PTI in bulls have been described in 2005 and 2014. ,19. In tVoges et al.  proposedThe localization of BVDV in the reproductive tract of Bull 2 was different to what had been observed for transiently infected bulls. BVDV RNA was only detected in the smaller of the testes. Relatively high levels of RNA and antigen were detected in the parenchyma of this infected testis and BVDV was also isolated in cell culture. This is in contrast to what had been observed for transiently infected bulls, where virus was not isolated from the testicular parenchyma. In this bull, trace levels of BVDV RNA were also detected in the epididymis but, unlike observations of transiently infected bulls, no antigen was detected. Unfortunately, the accessory glands of Bull 2 were not examined, preventing further comparisons with the observations made in transiently infected bulls where virus was only isolated from the accessory glands.The pathology and IHC staining of Bull 2 clearly demonstrated that the persistence of the BVDV infection was localized within the immune-privileged seminiferous tubule compartment. The bovine testis consists of two compartments, the peritubular compartment (Leydig cells and testicular macrophages) and the seminiferous tubule compartment (germ cells protected by Sertoli cells). The Sertoli cells span the basal to adluminal sections of the tubule and tight junctions between these cells form the blood–testes barrier . This baThe pattern of pathology detected in this testis shows a continuum of tubular degenerative change correlating closely with a progression of BVDV staining, suggesting that the infection in this case was confined to seminiferous tubules and chronicity resulted in the eventual destruction of the normal tubule structure. It is also noteworthy that the concentration of BVDV RNA was not uniform across the testis. Within one area of the testicular parenchyma, the qRT-PCR gave a Ct value of 37.3. This result indicates that the level of BVDV RNA present was very close to the limit of detection for this assay. Yet, in the other areas of the testicular parenchyma tested, the Ct values ranged from 23.9 to 28.0. These values are typically seen in the semen of PI cattle. We speculate that the infection in PTI remains largely hidden from the immune system and manages to smoulder within the seminiferous tubules for a variable amount of time. The case of the bull from which the testes were collected at the abattoir supports this notion as there was a lack of interstitial or tubular inflammatory cellular infiltrates in response to the widespread presence of BVDV within the tubules, even in areas where BVDV tubule staining appears to extend superficially into the subjacent thickened peritubular mesenchyme of intensely stained tubules This latter observation may be a by-product of excessive antigen accumulation in these tubules, or perhaps the peritubular fibrosis assisted to shelter the infection from the peritubular compartment immune response. We speculate that the destruction of the tubules contributed to the small overall size of this testis. It was not possible to determine if the uninfected testis was ever infected, though no histological signs of past infection were detected. BVDV RNA was only detected in one of the early semen collections in 2012. It is not clear if BVDV RNA levels were too low to detect in these collections, or if there was deterioration in the RNA over the six years between when the RNA was shed and when it was tested by qRT-PCR. Additionally, the final two semen collections for this bull gave negative results in the qRT-PCR. Nevertheless, the ability to isolate virus from the testis of this bull 73 months after the initial infection demonstrates the capacity of the infection to persist in the testis for long periods.The time course over which a PTI may be detected appears to range from a few months to many years. Eventually infection may be cleared, as appeared to occur in Bull 5 and as described by Newcomer et al., , or infeVirus isolation has been considered the gold standard for BVDV detection when screening semen for the presence of BVDV-1. However, the results of the current investigations demonstrate the benefits of using qRT-PCR, which offers both higher analytical sensitivity and speed for the detection of BVDV infection. It is important that qRT-PCR assays employ broadly reactive pan-Pestivirus primers and probes to ensure that the potential genetic variability of all strains of Pestiviruses are reliably detected . Many ofWith an increasing number of countries progressing towards freedom from BVDV, consideration may need to be given to screening of batches of stored semen. Most collections of semen from bulls that experience a PTI have very low levels of infectious virus and perhaps often at levels that would be lower than an infective dose following insemination. Nevertheless, this semen does present a biosecurity risk. Indeed, it had been shown that use of semen from the bull with the first recognized PTI was able to establish a systemic infection in an inseminated heifer , therebyIn conclusion, although prolonged or persistent testicular infections are a relatively rare event, the findings of this study emphasize the recommendations  for all


On January 7, 2019, the Oregon Public Health Division (OPHD) was contacted by a local health department regarding an Oregon teen who, on December 24, 2018, was bitten by a macaque monkey  in a pubMacacine herpesvirus 1 (B virus). This virus, commonly found in macaques,On January 5, 2019, the patient left Thailand and was evaluated by a physician in Oregon on January 7. The physician contacted the local health department, seeking guidance about when to administer the final dose of rabies vaccine. Upon learning about the macaque bite, the local health department contacted OPHD, where staff members expressed concern about possible exposure to OPHD recommended that if illness compatible with B virus infection developed  the patient should seek medical evaluation, and the provider should notify NBVRC immediately. On January 8, 2019, the patient received the final dose of rabies vaccine. Per recommendations for persons possibly exposed to B virus, serum specimens were collected at that visit and 3 weeks laterFollowing an initial exposure to B virus, the peripheral viral load can be insufficient to stimulate an immune response and can result in negative tests for antibodies against B virus. B virus can migrate to the dorsal ganglion and cause infection years later . Because B virus can establish a lifelong latent infection with possible subsequent illness . Nearly all documented B virus infections in humans involved exposures in laboratories or animal facilities (Rabies from nonhuman primate bites is uncommon because primates are not primary rabies reservoirs. Nonetheless, rabies postexposure prophylaxis for victims of nonhuman primate bites in countries where rabies is enzootic should be considered (