Porcine Epidemic Diarrhea Virus, Surrogate for Coronavirus Decay Measurement in French Coastal Waters and Contribution to Coronavirus Risk Evaluation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in infected patients mainly displays pulmonary and oronasal tropism; however, the presence of the virus has also been demonstrated in the stools of patients and consequently in wastewater treatment plant effluents, raising the question of the potential risk of environmental contamination (such as seawater contamination) through inadequately treated wastewater spillover into surface or coastal waters even if the environmental detection of viral RNA alone does not substantiate risk of infection. Therefore, here, we decided to experimentally evaluate the persistence of the porcine epidemic diarrhea virus (PEDv), considered as a coronavirus representative model, in the coastal environment of France. Coastal seawater was collected, sterile-filtered, and inoculated with PEDv before incubation for 0 to 4 weeks at four temperatures representative of those measured along the French coasts throughout the year (4, 8, 15, and 24°C). The decay rate of PEDv was determined using mathematical modeling and was used to determine the half-life of the virus along the French coast in accordance with temperatures from 2000 to 2021. We experimentally observed an inverse correlation between seawater temperature and the persistence of infectious viruses in seawater and confirm that the risk of transmission of infectious viruses from contaminated stool in wastewater to seawater during recreational practices is very limited. The present work represents a good model to assess the persistence of coronaviruses in coastal environments and contributes to risk evaluation, not only for SARS-CoV-2 persistence, but also for other coronaviruses, specifically enteric coronaviruses from livestock. IMPORTANCE The present work addresses the question of the persistence of coronavirus in marine environments because SARS-CoV-2 is regularly detected in wastewater treatment plants, and the coastal environment, subjected to increasing anthropogenic pressure and the final receiver of surface waters and sometimes insufficiently depurated wastewater, is particularly at risk. The problem also arises in the possibility of soil contamination by CoV from animals, especially livestock, during manure application, where, by soil impregnation and runoff, these viruses can end up in seawater. Our findings are of interest to researchers and authorities seeking to monitor coronaviruses in the environment, either in tourist areas or in regions of the world where centralized systems for wastewater treatment are not implemented, and more broadly, to the scientific community involved in "One Health" approaches.

Can shellfish be used to monitor SARS-CoV-2 in the coastal environment?

The emergence and worldwide spread of SARS-CoV-2 raises new concerns and challenges regarding possible environmental contamination by this virus through spillover of human sewage, where it has been detected. The coastal environment, under increasing anthropogenic pressure, is subjected to contamination by a large number of human viruses from sewage, most of them being non-enveloped viruses like norovirus. When reaching coastal waters, they can be bio-accumulated by filter-feeding shellfish species such as oysters. Methods to detect this viral contamination were set up for the detection of non-enveloped enteric viruses, and may need optimization to accommodate enveloped viruses like coronaviruses (CoV). Here, we aimed at assessing methods for the detection of CoV, including SARS-CoV-2, in the coastal environment and testing the possibility that SARS-CoV-2 can contaminate oysters, to monitor the contamination of French shores by SARS-CoV-2 using both seawater and shellfish. Using the porcine epidemic diarrhea virus (PEDV), a CoV, as surrogate for SARS-CoV-2, and Tulane virus, as surrogate for non-enveloped viruses such as norovirus, we assessed and selected methods to detect CoV in seawater and shellfish. Seawater-based methods showed variable and low yields for PEDV. In shellfish, the current norm for norovirus detection was applicable to CoV detection. Both PEDV and heat-inactivated SARS-CoV-2 could contaminate oysters in laboratory settings, with a lower efficiency than a calicivirus used as control. Finally, we applied our methods to seawater and shellfish samples collected from April to August 2020 in France, where we could detect the presence of human norovirus, a marker of human fecal contamination, but not SARS-CoV-2. Together, our results validate methods for the detection of CoV in the coastal environment, including the use of shellfish as sentinels of the microbial quality of their environment, and suggest that SARS-CoV-2 did not contaminate the French shores during the summer season.

Porcine epidemic diarrhea virus: Viral RNA detection and quantification using a validated one-step real time RT-PCR.

Since 2014, porcine epidemic diarrhea virus (PEDV) has reemerged in Europe. RT-PCR methods have been described for the detection of PEDV, but none have been validated according to a norm. In this study we described the development and validation of a SYBR™ Green one-step RT-qPCR according to the French norm NF U47-600, for the detection and quantification of PEDV viral RNA. The method was validated from sample preparation (feces or jejunum) through to nucleic acid extraction and RT-qPCR detection. Specificity and sensitivity, limit of detection (LoD), limit of quantification (LQ), linearity, intra and inter assay variability were evaluated using transcribed RNA and fecal and jejunum matrices spiked with virus. The analytical and diagnostic specificities and sensitivities of this RT-qPCR were 100% in this study. A LoD of 50 genome copies/5 µl of extract from fecal matrices spiked with virus or RNA transcript and 100 genome copies/5 µl of extract from jejunum matrices spiked with virus were obtained. The Lower LQ (LLQ) was 100 genome copies/5 µl and the Upper LQ (ULQ) 108 copies/5 µl. This method is the first, validated according a norm for PEDV and may serve as a global reference method to harmonize detection and quantification of PEDV viral RNA in both field and experimental settings.

PCV2 co-infection does not impact PRRSV MLV1 safety but enhances virulence of a PRRSV MLV1-like strain in infected SPF pigs.

Co-infection by a type 1 modified live vaccine-like strain (MLV1-like) of porcine reproductive and respiratory syndrome virus (PRRSV) and a type 2 porcine circovirus (PCV2) was identified on a French pig farm with post-weaning multisystemic wasting syndrome (PMWS). An in vivo experiment was set up to characterize the virulence level of the MLV1-like strain compared with the parental MLV1 strain, and to assess the impact of PCV2 co-infection on the pathogenicity of both PRRSV strains. Six groups of six pigs each were inoculated only with either one of the two PRRSV strains or with PCV2, or co-inoculated with PCV2 and MLV1 or PCV2 and MLV1-like strains. Six contact pigs were added to each inoculated group to assess viral transmission. The animals were monitored daily for 35 days post-inoculation for clinical symptoms. Blood and nasal swabs were sampled twice a week, and tissue samples were collected during necropsy for viral quantification. Compared to MLV1-infected pigs, animals infected with the MLV1-like strain had increased PRRSV viremia and nasal shedding, a higher viral load in the tonsils, and lymph node hypertrophy at microscopic level. PCV2 co-infection did not influence clinical, virologic or transmission parameters for MLV1, but co-infected MLV1-like/PCV2 pigs had the most severe lung lesions, the highest viremia in contact animals and the highest transmission rate. Our study demonstrated that the MLV1 strain tested was safe when co-inoculated with PCV2 in piglets. However, co-infection by the MLV1-like strain and PCV2 resulted in increased virulence compared with that due to a single infection.

Limited shedding of an S-InDel strain of porcine epidemic diarrhea virus (PEDV) in semen and questions regarding the infectivity of the detected virus.

PEDV is mainly transmitted by the oro-fecal route although PEDV shedding in semen has already been shown for an S-non-InDel PEDV strain infection. The aim of this study was to determine if PEDV can be shed in semen from SPF (specific pathogens free) boars infected by a French S-InDel PEDV strain (PEDV/FR/001/2014) and in case of positive semen to determine the infectivity of that semen. Both infected boars had diarrhea after inoculation and shed virus in feces. PEDV genome was also detected by RT-qPCR in the sperm-rich fraction of semen (6.94 × 103 and 4.73 × 103 genomic copies/mL) from the two boars infected with the S-InDel PEDV strain but only once at 7DPI. In addition, PEDV RNA in Peyer's patches and in mesenteric lymph nodes was also present for the two inoculated boars. The PEDV positive semen (S-non-InDel and S-InDel) sampled during a previous trial and in this boar trial were inoculated to six SPF weaned pigs. The inoculated piglets did not seroconvert and did not shed virus throughout the duration of the study except for one pig at 18 DPI. But, PEDV could be detected in intestinal tissues such as duodenum, jejunum and jejunum Peyer's patches by RT-qPCR except for one pig. Even if PEDV genome has been detected in semen, experimental infection of piglets with positive semen failed to conclude to the infectivity of the detected PEDV.

Lessons learnt from a porcine epidemic diarrhea (PED) case in France in 2014: Descriptive epidemiology and control measures implemented.

An acute epidemic of porcine epidemic diarrhea (PED) has affected the USA since 2013 and spread all around the world. In France, the immune status of the pig population against PED virus (PEDV) was expected to be low due to the absence of circulation of the virus since the 80's and a compulsory notification of PED was set up in 2014. Here, we reported the first case of a PED outbreak in December 2014 in the North of France after a long absence of the disease, the monitoring of the excretion and the control measure implementation. The isolated strain in France in December 2014 was a PEDV "S-InDel" strain which was close to the "S-InDel" German PEDV strain isolated in May 2014. The individual shedding duration of PEDV in feces was estimated around 20 days for pigs of different ages. Biosecurity measures implemented allowed the limitation of PEDV spread to fattening and farrowing rooms without dissemination to the nursery block. Using strict biosecurity measures, direct shipment of infected fatteners to the slaughterhouse, strict decontamination protocols with a quarantine of 6 weeks for replacement gilts without voluntary contamination helped PEDV fade out within the herd and avoided the spread to other herds. PEDV presence in manure was investigated as well as the inactivation treatment of the virus present in the liquid manure. An increase to a pH 12 of liquid manure by liming led to the absence of PEDV detection by RT-PCR after seven days.

Better horizontal transmission of a US non-InDel strain compared with a French InDel strain of porcine epidemic diarrhoea virus.

From the severe porcine epidemic diarrhoea (PED) epidemics that struck in 2013 in the United States of America and other countries of North and South America, two types of porcine epidemic diarrhoea virus (PEDV) were isolated, namely the InDel and the non-InDel strains. They are differentiated by insertions/deletions in the S1 nucleotide sequence of the S gene, and differences in virulence were observed from the clinical cases. In 2014, a PED outbreak occurred in a pig farm in France, from which an InDel strain was isolated. This study aimed at comparing, under experimental conditions, the pathogenicity and the direct and indirect transmissions between a non-InDel strain isolated from a PED-affected piglet in 2014 in the USA and the French InDel strain. All infected pigs showed clinical signs with the non-InDel strain although only the inoculated and direct contact pigs showed clinical signs in the InDel strain group. Although viral RNA was detected in air samples with both strains, the indirect contact pigs remained free from infection with the InDel strain in contrast to the non-InDel group in which airborne transmission occurred in the indirect contact pigs. All infected pigs shed virus in faeces regardless of PEDV strain with 9 of 30 pigs showing intermittent faecal shedding. The transmission rate by direct contact was found to be 2.17-fold higher than the non-InDel strain compared with the InDel. In conclusion, the InDel strain was less pathogenic than the non-InDel strain in our experimental conditions. The transmission route differed between the two strains. Direct contact was the main transmission route for the InDel strain, although the non-InDel strain was transmitted through direct contact and indirectly through the air.

Evidence of porcine epidemic diarrhea virus (PEDV) shedding in semen from infected specific pathogen-free boars.

In 2013, PED emerged for the first time in the United States (US). The porcine epidemic diarrhea virus (PEDV) spread quickly throughout North America. Infection with PEDV causes watery diarrhea and up to 100% mortality in piglets, particularly for highly pathogenic non-InDel strains circulating in the US. PEDV is mainly transmitted by the fecal-oral route. Transmission via the venereal route has been suspected but not previously investigated. The aim of the study was to determine if PEDV could be detected in semen from infected specific pathogen-free (SPF) boars inoculated with a PEDV US non-InDel strain suggesting venereal transmission may occur. Two boars orally inoculated with PEDV showed clinical signs and virus shedding in feces. Transient presence of the PEDV genome was detected by RT-qPCR in the seminal (5.06 × 102 to 2.44 × 103 genomic copies/mL) and sperm-rich fraction of semen (5.64 × 102 to 3.40 × 104 genomic copies/mL) and a longer duration of viral shedding was observed in the sperm-rich fraction. The evidence of PEDV shedding in semen raises new questions in term of disease spread within the pig population with the use of potentially contaminated semen.

Promising new vaccine candidates against Campylobacter in broilers.

Campylobacter is the leading cause of human bacterial gastroenteritis in the European Union. Birds represent the main reservoir of the bacteria, and human campylobacteriosis mainly occurs after consuming and/or handling poultry meat. Reducing avian intestinal Campylobacter loads should impact the incidence of human diseases. At the primary production level, several measures have been identified to reach this goal, including vaccination of poultry. Despite many studies, however, no efficient vaccine is currently available. We have recently identified new vaccine candidates using the reverse vaccinology strategy. This study assessed the in vivo immune and protective potential of six newly-identified vaccine antigens. Among the candidates tested on Ross broiler chickens, four (YP_001000437.1, YP_001000562.1, YP_999817.1, and YP_999838.1) significantly reduced cecal Campylobacter loads by between 2 and 4.2 log10 CFU/g, with the concomitant development of a specific humoral immune response. In a second trial, cecal load reductions results were not statistically confirmed despite the induction of a strong immune response. These vaccine candidates need to be further investigated since they present promising features.

The expression level of gC1qR is down regulated at the early time of infection with porcine circovirus of type 2 (PCV-2) and gC1qR interacts differently with the Cap proteins of porcine circoviruses.

Porcine circoviruses (PCV) are small, non-enveloped single-stranded DNA-viruses. Porcine circovirus type 2 (PCV-2) is the causal agent of post-weaning multisystemic wasting syndrome (PMWS) whereas porcine circovirus of type 1 (PCV-1) is non- pathogenic. gC1qR is a membrane-located receptor of the complement protein subunit C1q and interacts with PCV capsid proteins. The mechanisms associated with the triggering of PMWS are not well known and gC1qR may have a role in the life cycle and eventually in the pathogenicity of PCV. The objectives of this study were to determine the level of expression of gC1qR during early PCV-2 infection, to determine the region of PCV-2 capsid protein (Cap) required for the interaction with gC1qR and to evaluate the interaction of gC1qR with Cap proteins of different PCV strains. The results indicate that gC1qR transcripts are downregulated in the tonsils and the tracheo-bronchial lymph nodes of piglets infected by PCV-2 at the early time of the infection. The N-terminal amino acids (a.a. 1-59) of PCV-2b Cap, an arginine rich region, are involved in the interaction with gC1qR. Porcine gC1qR interacts with Cap proteins of two pathogenic viral strains, PCV-2a and PCV-2b, while interaction has been observed with only one Cap protein of two investigated strains of PCV-1. The amino acids 30 and 49 of PCV-1Cap, solely, were not responsible of the difference of interaction observed. We have also shown that gC1qR interacts strongly with PCV-2Caps and PCV-1 GER Cap. This result suggests that the different interaction of gC1qR with PCV Cap proteins may have an impact on the pathogenicity of the PCV.

Complete genome sequence of a porcine epidemic diarrhea s gene indel strain isolated in france in december 2014.

We report the first and only case of a porcine epidemic diarrhea (PED) outbreak occurring in December 2014 in northern France, and we show using the full-length genome sequence of the French PED virus (PEDV) isolate that it was a PEDV indel strain close to German PEDV strains recently isolated.

Evaluation of the transmission of porcine circovirus type 2 (PCV-2) genogroups a and b with semen from infected specific-pathogen-free boars.

The porcine circovirus type 2 (PCV-2) is associated with several diseases including reproductive failure. This syndrome has been experimentally reproduced twice with two PCV-2 isolates representative of each major PCV-2 genogroup, i.e. PCV-2a and PCV-2b (Cariolet et al., 2002; Rose et al., 2007). In these two previous studies, the sows were infected by intra-uterine inoculation at insemination with 10(4.3) and 10(3.18) TCID(50) of PCV-2a and PCV-2b, respectively, corresponding to 1.2 × 10(11) and 3 × 10(10) genome copies, respectively. The aim of this present study was to quantify viral shedding in semen from specific-pathogen-free (SPF) boars infected with isolates from the two major PCV-2 genogroups a and b. We studied the transmission of the PCV-2 virus through contaminated semen to SPF sows and their offspring. The four inoculated boars developed sub-clinical PCV-2 infections and PCV-2 genomes were occasionally detected in semen after nasal infection of boars, with up to 1.2 × 10(6)copies/mL in the sperm-rich fraction. When PCV-2-contaminated semen was inoculated in SPF sows at artificial insemination, the sows and their offspring did not show any signs of PCV-2 infection or PCV-2 antibodies or genomes. In the present study, sows were inoculated with a maximal dose of 1.7 × 10(7) viral genome copies, which is lower than the genomic loads (i.e. 1.2 × 10(11) and 3 × 10(10) genome copies) that have been shown to induce reproductive troubles in intra-uterine inoculated sows. Our results together with the previous experiment findings suggest that PCV-2-induced reproductive disorders depend on the infectious dose inoculated to sows by the intra-uterine route.

Naked PCV-2 cloned genomic DNA is infectious by mucosal (intratracheal or oro-nasal) inoculation.

Porcine circovirus type 2 (PCV-2) is involved in several diseases named porcine circovirus-associated diseases and is transmitted by oro-faecal route. In this study we inoculated porcine-circovirus free piglets by mucosal routes (intratracheal or oro-nasal routes) with a plasmid carrying two copies of PCV-2 genomic DNA and compared the results to the intramuscular route. We observed that this PCV-2 naked DNA serves as template for viral replication and infectious PCV-2 particles are detected in the whole body after parenteral (intramuscular) or mucosal (intratracheal or oro-nasal) delivery. These results suggest that PCV-2 genome could play a role in in vivo transmission.