A study of the presence of human viruses (adenoviruses, enteroviruses, and hepatitis A viruses [HAVs]) in environmental and shellfish samples was carried out by applying DNA and cDNA amplification techniques by PCR. The detection of human adenoviruses by PCR was also examined as a potential molecular test to monitor viral pollution. The samples studied were urban and slaughterhouse sewage, river water, seawater, and shellfish. Enteroviruses were quantified by PFU in Buffalo green monkey kidney cells and fecal coliforms and phages of Bacteroides fragilis HSP40 were also evaluated in some of the samples. The amplification of viral DNA and cDNA has shown a high prevalence of human viruses that would not be detected by the use of classical techniques, such as the quantification of PFU in cell lines. The results of the analysis of slaughterhouse sewage samples together with the test of farm animal feces indicate that the adenoviruses and the HAVs detected in the environment are mostly of human origin. A significative correlation between the detection of human viruses by PCR and the values of bacteriophages of B. fragilis HSP40 in urban raw sewage was observed. Human adenoviruses were the viruses most frequently detected throughout the year, and all the samples that were positive for enteroviruses or HAVs were also positive for human adenoviruses. The results suggest that the detection of adenoviruses by PCR could be used as an index of the presence of human viruses in the environment where a molecular index is acceptable.

African swine fever is a highly contagious viral disease of mandatory declaration to the World Organization for Animal Health (OIE). The lack of available vaccines makes its control difficult; thus, African swine fever virus (ASFV) represents a major threat to the swine industry. Inactivated vaccines do not confer solid protection against ASFV. Conversely, live attenuated viruses (LAV), either naturally isolated or obtained by genetic manipulation, have demonstrated reliable protection against homologous ASFV strains, although little or no protection has been demonstrated against heterologous viruses. Safety concerns are a major issue for the use of ASFV attenuated vaccine candidates and have hampered their implementation in the field so far. While trying to develop safer and efficient ASFV vaccines, we found that the deletion of the viral CD2v (EP402R) gene highly attenuated the virulent BA71 strainin vivo. Inoculation of pigs with the deletion mutant virus BA71ΔCD2 conferred protection not only against lethal challenge with the parental BA71 but also against the heterologous E75 (both genotype I strains). The protection induced was dose dependent, and the cross-protection observedin vivocorrelated with the ability of BA71ΔCD2 to induce specific CD8⁺T cells capable of recognizing both BA71 and E75 virusesin vitro. Interestingly, 100% of the pigs immunized with BA71ΔCD2 also survived lethal challenge with Georgia 2007/1, the genotype II strain of ASFV currently circulating in continental Europe. These results open new avenues to design ASFV cross-protective vaccines, essential to fight ASFV in areas where the virus is endemic and where multiple viruses are circulating.IMPORTANCEAfrican swine fever virus (ASFV) remains enzootic in most countries of Sub-Saharan Africa, today representing a major threat for the development of their swine industry. The uncontrolled presence of ASFV has favored its periodic exportation to other countries, the last event being in Georgia in 2007. Since then, ASFV has spread toward neighboring countries, reaching the European Union's east border in 2014. The lack of available vaccines against ASFV makes its control difficult; so far, only live attenuated viruses have demonstrated solid protection against homologous experimental challenges, but they have failed at inducing solid cross-protective immunity against heterologous viruses. Here we describe a new LAV candidate with unique cross-protective abilities: BA71ΔCD2. Inoculation of BA71ΔCD2 protected pigs not only against experimental challenge with BA71, the virulent parental strain, but also against heterologous viruses, including Georgia 2007/1, the genotype II strain of ASFV currently circulating in Eastern Europe.

To determine the prevalence of Hepatitis E virus (HEV) in industrialized nations, we analyzed the excretion of HEV strains by the populations of Spain, France, Greece, Sweden, and the United States. Twenty of 46 (43.5%) urban sewage samples collected in Barcelona from 1994 to 2002 tested positive for HEV. We identified 15 HEV strains, which were similar to two HEV isolates previously described in Barcelona in clinical samples and to strains from diverse geographic HEV-nonendemic areas. We also identified two HEV strains in sewage samples from Washington, D.C., and Nancy, France; these samples were also positive for Hepatitis A virus. In addition, we studied the role of pigs as a reservoir for HEV and identified one new swine HEV strain. Our results suggest that HEV may be more prevalent than previously considered in industrialized countries and that variants of the virus circulate simultaneously in one region.

This is the first description, to our knowledge, of the distribution of human polyomavirus and simian virus 40 (SV40) in urban sewage. Using a nested-PCR procedure, we report the detection of human polyomaviruses JC virus (JCV) and BK virus (BKV) but not SV40 in a high percentage of urban sewage samples obtained from widely divergent geographical areas in Europe and Africa. For a total of 28 samples analyzed, JCV was detected in 26, BKV was detected in 22, and none was positive for SV40. All geographical areas showed a high prevalence of these viruses with mean estimated values of JC viral particles per ml on the order of 10 ³ in Barcelona (Spain) and Nancy (France) and 10 ² in Pretoria (South Africa) and Umeå (Sweden) and mean values of BK viral particles on the order of 10 ² in Pretoria and Barcelona and 10 ¹ in Nancy and Umeå. This compares with estimated mean values of 10 ² to 10 ³ for human adenovirus that was evaluated as a control. Nucleotide sequence analysis of the amplified DNA from some of the samples is also presented and represents the sequence of the most abundant JC and BK viral strains in these samples. The nucleotide sequence of the JCV detected was also analyzed in a phylogenetic study and for genomic characterization in the regulatory region. This study has shown that human polyomaviruses are spread in high concentrations in the sewage of different geographical areas and are present in contaminated environments. The frequency and concentration of JCV detected in the environment and the absence of described animal hosts suggest that JCV may be useful as a marker for fecal pollution of anthropogenic origin. The results also support the idea previously described that the strains of JCV are closely related to the ethnic origin of the population studied. The procedure applied should also be useful in future studies of population patterns of viral excretion and as a tool in epidemiological studies for the detection of changes in the prevalence of specific viral pathogens.

African swine fever is one of the most devastating pig diseases, against which there is no vaccine available. Recent work from our laboratory has demonstrated the protective potential of DNA vaccines encoding three African swine fever viral antigens (p54, p30, and the hemagglutinin extracellular domain) fused to ubiquitin. Partial protection was afforded in the absence of detectable antibodies prior to virus challenge, and survival correlated with the presence of a large number of hemagglutinin-specific CD8 ⁺ T cells in blood. Aiming to demonstrate the presence of additional CD8 ⁺ T-cell determinants with protective potential, an expression library containing more than 4,000 individual plasmid clones was constructed, each one randomly containing a Sau3AI restriction fragment of the viral genome (p54, p30, and hemagglutinin open reading frames [ORFs] excluded) fused to ubiquitin. Immunization of farm pigs with the expression library yielded 60% protection against lethal challenge with the virulent E75 strain. These results were further confirmed by using specific-pathogen-free pigs after challenging them with 10 ⁴ hemadsorbing units (HAU) of the cell culture-adapted strain E75CV1. On this occasion, 50% of the vaccinated pigs survived the lethal challenge, and 2 out of the 8 immunized pigs showed no viremia or viral excretion at any time postinfection. In all cases, protection was afforded in the absence of detectable specific antibodies prior to challenge and correlated with the detection of specific T-cell responses at the time of sacrifice. In summary, our results clearly demonstrate the presence of additional protective determinants within the African swine fever virus (ASFV) genome and open up the possibility for their future identification. IMPORTANCE African swine fever is a highly contagious disease of domestic and wild pigs that is endemic in many sub-Saharan countries, where it causes important economic losses and is currently in continuous expansion across Europe. Unfortunately, there is no treatment nor an available vaccine. Early attempts using attenuated vaccines demonstrated their potential to protect pigs against experimental infection. However, their use in the field remains controversial due to safety issues. Although inactive and subunit vaccines did not confer solid protection against experimental ASFV infection, our DNA vaccination results have generated new expectations, confirming the key role of T-cell responses in protection and the existence of multiple ASFV antigens with protective potential, more of which are currently being identified. Thus, the future might bring complex and safe formulations containing more than a single viral determinant to obtain broadly protective vaccines. We believe that obtaining the optimal vaccine formulation it is just a matter of time, investment, and willingness.