Selection of viral variants with enhanced transmission and reduced neutralization susceptibility alongside lateral introductions may explain the persistence of porcine reproductive and respiratory syndrome virus in vaccinated breeding herds.

This study investigates the long-term evolutionary dynamics of porcine reproductive and respiratory syndrome virus (PRRSV-1) in an endemically infected and vaccinated pig herd. Over a one year and a half period, piglets from seven farrowing batches in a 300-sow PRRSV-vaccinated farm were monitored from birth to nine weeks of age by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Eighty-five PRRSV-positive samples were subjected to whole genome sequencing (Illumina Miseq), and 251 samples to open reading frame 5 (ORF5) sequencing. Farm-specific PRRSV variants' impact on anti-PRRSV antibodies was evaluated using enzyme-linked immunosorbent and neutralizing antibody assays. The replication kinetics and cytokine inhibition capabilities (IFN-α and TNF-α) of these variants were assessed in porcine alveolar macrophages. The study revealed fluctuating PRRSV-1 incidences in farrowing units and nurseries, attributed to two key evolutionary events: an escape variant emergence and a lateral introduction of a new strain. Initially, strain 1 variant α was swiftly replaced within weeks by variant 1ß (99.5 per cent genomic similarity), with twenty-five amino acid mutations, primarily in nsp1α, GP2, GP3, and GP5, including an additional glycosylation site and a deletion downstream the neutralization epitope of GP5. This shift to 1ß correlated with increased incidence in nurseries and higher viral loads, with sera from 1α-exposed animals showing reduced neutralization against 1ß. Consistently for in vitro assays, variant 1ß demonstrated enhanced replication in porcine alveolar macrophages but no difference regarding IFN-α or TNF-α responses. Later, a new strain (strain 2, 83.3 per cent similarity to strain 1) emerged and led to incidence resurgence because of the low cross reactivity with the previous antibodies. The study highlights PRRSV's rapid adaptability and challenges in controlling its spread, underscoring the necessity for more effective vaccines and eradication approaches.

Study of Canine Distemper Virus Presence in Catalonia's Wild Carnivores through H Gene Amplification and Sequencing.

Canine distemper virus (CDV) is recognised worldwide as an important pathogen in both domestic and wild carnivores. Few data are available on its impact and spread on the wildlife/wildlife-domestic animal-environment interface. This study, aimed at developing a conservation-oriented control strategy, analysed 89 sick or deceased animals from 2019 to 2023 at the Wildlife Rehabilitation Centre in Torreferrussa. RT-PCR and sequencing of the partial H gene were used to detect and analyse CDV in tissues. The total positive percentage was 20.22% (18/89), comprising 13 red foxes (44.8%), 4 European badgers (28.6%), and 1 American mink (4.5%), while 24 Eurasian otters tested negative. Phylogenetic analysis indicated that all of the CDV strains belong to the European lineage. Geographically distant individuals and different species shared the same viral strain, suggesting a strong capacity of CDV for interspecies and long-distance transmission. This calls for further research, particularly focusing on potential impacts of CDV on endangered carnivores.

Levels of neutralizing antibodies against resident farm strain or vaccine strain are not indicators of protection against PRRSV-1 vertical transmission under farm conditions.

BACKGROUND: Vertical transmission is key for the maintenance of porcine reproductive and respiratory syndrome virus (PRRSV) infection. In vaccinated farms, vertical transmission can still occur despite sows having some level of immunity because of repeated vaccination or contact with the wild-type virus. The present study aimed to correlate the age of sows and the amplitude of neutralizing antibodies (Nab) (heterologous neutralization) with PRRSV-1 vertical transmission (VT). For this purpose, umbilical cords of 1,554 newborns (corresponding to 250 litters) were tested for PRRSV by RT-PCR in two PRRSV-unstable vaccinated farms. In parallel, the sows were bled after farrowing and the levels of antibodies were determined by ELISA and by the viral neutralization test against the vaccine virus, the virus circulating in the farm, and other unrelated contemporary PRRSV-1 strains. The relationship between the parity and the probability of delivering infected piglets and the presence of broadly Nabs examined. RESULTS: The proportion of VT events in the two examined farms ranged from 18.9% to 23.0%. Young sows (parity 1-2) were 1.7 times more likely to have VT than older sows (p < 0.05). Despite higher ELISA S/P antibody ratios in younger sows (p < 0.05), NAb against the resident farm strain were at a similar level between sows delivering infected and healthy piglets regardless of age, mostly with low titers (2-3 log2). The titers of NAb against the vaccine virus were also low, and no correlations with VT were observed. When a panel of another 4 strains (1 isolated in the 1990s, and 3 contemporary strains) were used for the neutralization test, most sow sera were not capable of neutralizing the contemporary strains. CONCLUSIONS: Titers of NAb could not be correlated with the occurrence of PRRSV VT. The amplitude of NAb present in most vaccinated sows is limited with a considerable proportion unresponsive regarding NAb production.

High levels of maternally derived antibodies do not significantly interfere with the development of humoral and cell-mediated responses to Porcine circovirus 2 after intradermal vaccination.

BACKGROUND: Vaccination of pigs against PCV2 is usually performed around weaning when animals still have maternally derived antibodies (MDA). The present study aimed to assess the possible interference of MDA in the development of the PCV2-specific immune response after vaccination of commercial weaners. For this purpose, a PRRS-negative 600-sow farrow-to-finish farm was selected. Half of the sows were vaccinated and revaccinated with Porcilis® PCV ID against PCV2 7 and 3 weeks before farrowing. After farrowing, piglets were tested by AlphaLisa to select 72 animals with high and low levels of MDA. Groups were further subdivided and vaccinated intradermally with Porcilis® PCV ID at 21 or 28 days of age. Unvaccinated controls were also included. Animals were followed afterward for 42 days to examine the development of PCV2-specific antibodies and interferon-γ secreting cells (IFN-γ SC). RESULTS: The average titres of antibodies of the groups vaccinated in the presence of low or high MDA levels were similar at 28 and 42 days post-vaccination while in the controls the titres declined throughout the observation period. Results of vaccinating at 21 or 28 days of age were equivalent with regard to antibody development. Regarding the IFN-γ SC, vaccinated animals produced significant frequencies of IFN-γ SC by day 28. Again, no differences were observed between the groups with high or low antibody levels. CONCLUSION: High levels of MDA did not interfere with the development of humoral and cell-mediated responses to Porcine circovirus 2 after intradermal vaccination at 21 or 28 days of age.

Detection and genetic characterization of enteric viruses in diarrhoea outbreaks from swine farms in Spain.

BACKGROUND: The aim of this work was to study the prevalence and distribution of Porcine astrovirus (PAstV), Porcine kobuvirus (PKoV), Porcine torovirus (PToV), Mammalian orthoreovirus (MRV) and Porcine mastadenovirus (PAdV) as well as their association with widely recognized virus that cause diarrhoea in swine such as coronavirus (CoVs) and rotavirus (RVs) in diarrhoea outbreaks from Spanish swine farms. Furthermore, a selection of the viral strains was genetically characterized. RESULTS: PAstV, PKoV, PToV, MRV and PAdV were frequently detected. Particularly, PAstV and PKoV were detected in almost 50% and 30% of the investigated farms, respectively, with an age-dependent distribution; PAstV was mainly detected in postweaning and fattening pigs, while PKoV was more frequent in sucking piglets. Viral co-infections were detected in almost half of the outbreaks, combining CoVs, RVs and the viruses studied, with a maximum of 5 different viral species reported in three investigated farms. Using a next generation sequencing approach, we obtained a total of 24 ARN viral genomes (> 90% genome sequence), characterizing for first time the full genome of circulating strains of PAstV2, PAstV4, PAstV5 and PToV on Spanish farms. Phylogenetic analyses showed that PAstV, PKoV and PToV from Spanish swine farms clustered together with isolates of the same viral species from neighboring pig producing countries. CONCLUSIONS: Although further studies to evaluate the role of these enteric viruses in diarrhoea outbreaks are required, their wide distribution and frequent association in co-infections cannot be disregard. Hence, their inclusion into routine diagnostic panels for diarrhoea in swine should be considered.

Infection dynamics, transmission, and evolution after an outbreak of porcine reproductive and respiratory syndrome virus.

The present study was aimed at describing the infection dynamics, transmission, and evolution of porcine reproductive and respiratory syndrome virus (PRRSV) after an outbreak in a 300-sow farrow-to-wean farm that was implementing a vaccination program. Three subsequent batches of piglets (9-11 litters/batch) were followed 1.5 (Batch 1), 8 (Batch 2), and 12 months after (Batch 3) from birth to 9 weeks of age. The RT-qPCR analysis showed that shortly after the outbreak (Batch 1), one third of sows were delivering infected piglets and the cumulative incidence reached 80% by 9 weeks of age. In contrast, in Batch 2, only 10% animals in total got infected in the same period. In Batch 3, 60% litters had born-infected animals and cumulative incidence rose to 78%. Higher viral genetic diversity was observed in Batch 1, with 4 viral clades circulating, of which 3 could be traced to vertical transmission events, suggesting the existence of founder viral variants. In Batch 3 though only one variant was found, distinguishable from those circulating previously, suggesting that a selection process had occurred. ELISA antibodies at 2 weeks of age were significantly higher in Batch 1 and 3 compared to Batch 2, while low levels of neutralizing antibodies were detected in either piglets or sows in all batches. In addition, some sows present in Batch 1 and 3 delivered infected piglets twice, and the offspring were devoid of neutralizing antibodies at 2 weeks of age. These results suggest that a high viral diversity was featured at the initial outbreak followed by a phase of limited circulation, but subsequently an escape variant emerged in the population causing a rebound of vertical transmission. The presence of unresponsive sows that had vertical transmission events could have contributed to the transmission. Moreover, the records of contacts between animals and the phylogenetic analyses allowed to trace back 87 and 47% of the transmission chains in Batch 1 and 3, respectively. Most animals transmitted the infection to 1-3 pen-mates, but super-spreaders were also identified. One animal that was born-viremic and persisted as viremic for the whole study period did not contribute to transmission.

Introduction of a PRRSV-1 strain of increased virulence in a pig production structure in Spain: virus evolution and impact on production.

BACKGROUND: A strain of Porcine reproductive and respiratory syndrome virus (PRRSV), showing characteristics of enhanced virulence, affected a pyramidal production system from Spain with 7600 sows in 4 genetic nuclei and 13,000 sows in multipliers. Different PRRSV strains circulating in this production system from December 2020 to October 2021 were detected and sequenced. The spread of each isolate was examined and their impact on health and production in three of the affected farms was evaluated. RESULTS: The newly emerged PRRSV isolate with enhanced virulence entered the system before the onset of the study (January 2020) and afterwards four significantly different clades were detected during the study period in different farms, probably because of independent introduction events. The diversification of the enhanced virulence strain was higher for those clades (substitution rates up to 1.1% nucleotides/year) compared to other PRRSV strains present in the production system (up to 0.17%), suggesting a faster spread and adaptation. The impact of the infection in the first affected farm was dramatic, with an average abortion rate above 27% during 17 weeks before returning to the baseline production. Fertile sow mortality reached 6.5% for 39 weeks. In two farms infected later by other clades of this enhanced virulence strain, the impact was less acute; despite the fact that for parameters such as the proportion of stillbirths or mummies, more than 10 months were needed to recover pre-outbreak values. In the examined nurseries, mortalities reached peaks between 28 and 50% and several months were needed to return to normality. CONCLUSION: Introduction of a PRRSV strain of enhanced virulence in a production system where several farms were previously positive for other PRRSV strains, resulted in a fast spread such as would be observed in naïve farms. The productive and health impact was very high taking several months to return to normality.

Persistence of low pathogenic avian influenza virus in artificial streams mimicking natural conditions of waterfowl habitats in the Mediterranean climate.

Avian influenza viruses (AIVs) can affect wildlife, poultry, and humans, so a One Health perspective is needed to optimize mitigation strategies. Migratory waterfowl globally spread AIVs over long distances. Therefore, the study of AIV persistence in waterfowl staging and breeding areas is key to understanding their transmission dynamics and optimizing management strategies. Here, we used artificial streams mimicking natural conditions of waterfowl habitats in the Mediterranean climate (day/night cycles of photosynthetic active radiation and temperature, low water velocity, and similar microbiome to lowland rivers and stagnant water bodies) and then manipulated temperature and sediment presence (i.e., 10-13 °C vs. 16-18 °C, and presence vs. absence of sediments). An H1N1 low pathogenic AIV (LPAIV) strain was spiked in the streams, and water and sediment samples were collected at different time points until 14 days post-spike to quantify viral RNA and detect infectious particles. Viral RNA was detected until the end of the experiment in both water and sediment samples. In water samples, we observed a significant combined effect of temperature and sediments in viral decay, with higher viral genome loads in colder streams without sediments. In sediment samples, we didn't observe any significant effect of temperature. In contrast to prior laboratory-controlled studies that detect longer persistence times, infectious H1N1 LPAIV was isolated in water samples till 2 days post-spike, and none beyond. Infectious H1N1 LPAIV wasn't isolated from any sediment sample. Our results suggest that slow flowing freshwater surface waters may provide conditions facilitating bird-to-bird transmission for a short period when water temperature are between 10 and 18 °C, though persistence for extended periods (e.g., weeks or months) may be less likely. We hypothesize that experiments simulating real environments, like the one described here, provide a more realistic approach for assessing environmental persistence of AIVs.

In the presence of non-neutralising maternally derived antibodies, intradermal and intramuscular vaccination with a modified live vaccine against porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) induce similar levels of neutralising antibodies or interferon-gamma secreting cells.

The purpose of this study was to compare the immune response generated by the intramuscular and the intradermal vaccination route against the porcine reproductive and respiratory syndrome virus (PRRSV). Piglets from a seronegative and a seropositive farm were selected (n = 28 piglets per farm), and each group was divided into two groups and vaccinated after weaning with modified live vaccine Unistrain® PRRS (Laboratorios Hipra Amer, Spain) by the intramuscular (IM) or the intradermic (ID) route. For the following 6 weeks, animals were weekly bled to assess the humoral response by PRRSV-specific antibody ELISA and viral neutralisation test. At 0-, 3-, 4- and 6 weeks post-vaccination, peripheral mononuclear blood cells (PBMC) from eight animals per group were recovered to analyse cellular response by IFN-γ ELISPOT and lymphoproliferation. Serum IL-12 was also quantified by ELISA. Seroconversion was first detected 14 days post-vaccination (dpv) for both IM and ID routes, and peaked at 35 dpv (both IM groups and ID seropositive) or 42 dpv (ID seronegative). At 3 weeks after vaccination, 6/27 (22.22%) animals from negative origin had not seroconverted, and neutralising titres were significantly lower at 35 dpv compared to the seropositive origin (mean log2 titres of 1.36 and 4.25 respectively) Also, it was 10 times more probable for them to have high levels of IL-12 a week after vaccination than for animals of seropositive origin. Cellular immune response analysed by lymphoproliferation and IFN-γ ELISPOT was already present at 21 dpv and until 42 dpv, with no significant differences between groups except for a higher lymphoproliferation at 35 dpv in the IM seropositive group (Kruskal-Wallis, p < 0.05). These results indicate that the intradermal route induces an immune response equivalent to the classical intramuscular route even in presence of non-neutralising maternal immunity, which in this study has proven to facilitate seroconversion after vaccination with an heterologous strain.

Characterization and cross-protection of experimental infections with SeCoV and two PEDV variants.

The aim of this study was to characterize the infection of weaned pigs with swine enteric coronavirus (SeCoV) - a chimeric virus most likely originated from a recombination event between porcine epidemic diarrhoea virus (PEDV) and transmissible gastroenteritis virus, or its mutant porcine respiratory coronavirus - and two PEDV G1b variants, including a recently described recombinant PEDV-SeCoV (rPEDV-SeCoV), as well as to determine the degree of cross-protection achieved against the rPEDV-SeCoV. For this purpose, forty-eight 4-week-old weaned pigs were randomly allocated into four groups of 12 animals. Piglets within each group were primary inoculated with one of the investigated viral strains (B: PEDV; C: SeCoV and D: rPEDV-SeCoV) or mock-inoculated (A), and exposed to rPEDV-SeCOV at day 20 post-infection; thus, group A was primary challenged (-/rPEDV-SeCoV), groups B and C were subjected to a heterologous re-challenge (PEDV/rPEDV-SeCoV and SeCoV/rPEDV-SeCoV, respectively), and group D to a homologous re-challenge (rPEDV-SeCoV/rPEDV-SeCoV), Clinical signs, viral shedding, microscopic lesions and specific humoral and cellular immune responses (IgG, IgA, neutralizing antibodies and IgA and IFN-γ-secreting cells) were monitored. After primo-infection, all three viral strains induced an undistinguishable mild-to-moderate clinical disease with diarrhoea as the main sign and villus shortening lesions in the small intestine. In homologous re-challenged pigs, no clinical signs or lesions were observed, and viral shedding was only detected in a single animal. This fact may be explained by the significant high level of rPEDV-SeCoV-specific neutralizing antibodies found in these pigs before the challenge. In contrast, prior exposure to a different PEDV G1b variant or SeCoV only provided partial cross-protection, allowing rPEDV-SeCoV replication and shedding in faeces.

Description of a New Clade within Subtype 1 of Betaarterivirus suid 1 Causing Severe Outbreaks in Spain.

This report describes 28 genome sequences from a new clade within subtype 1 of Betaarterivirus suid 1, formerly known as porcine reproductive and respiratory syndrome virus 1. All share a potential recombinant pattern, with a highly pathogenic Italian strain as the putative major parental sequence and three other possible parents.

Special Issue "Viral Shedding and Transmission in Zoonotic Diseases".

The papers published in this Special Issue represent only a glimpse of the vast diversity of viral infectious diseases, and the complexity of their interactions with the host, that have an impact on human and animal health [...].

Corrigendum: Dual Host and Pathogen RNA-Seq Analysis Unravels Chicken Genes Potentially Involved in Resistance to Highly Pathogenic Avian Influenza Virus Infection.

[This corrects the article DOI: 10.3389/fimmu.2021.800188.].

The use of a whole inactivated PRRS virus vaccine administered in sows and impact on maternally derived immunity and timing of PRRS virus infection in piglets.

Background: Porcine reproductive and respiratory syndrome virus (PRRSV) vaccination is usually based on administering periodically PRRS modified live virus (MLV) in sows throughout their life. Using this schedule, transfer of maternally derived antibodies to the offspring is limited. The aim of the present study was to test the concept of priming with an MLV and boosting with a commercial inactivated virus vaccine in sows to reduce PRRSV incidence and improve productivity. Methods: On two farms, all the sows were vaccinated with a MLV vaccine at week 8 of gestation. Then two groups were designated, one group was re-vaccinated in the third week prior to farrowing and using a commercial inactivated vaccine (the PG group). The second group was the control group (C). Assays for PRRSV infection and productive parameters were evaluated. Results: For both farms, the incidence of PRRSV was lower at 6 weeks of age in PG than in C (p < 0.05). At weaning the proportion of PRRSV seropositive piglets was higher for PG as well (p < 0.05). The litters from C sows from both farms showed a higher pre-weaning mortality (odds ratio, C vs. PG = 1.18 ± 0.09; p < 0.05). Conclusions: Administration of the vaccine in sows before farrowing was safe and associated with reduced incidence of PRRSV in piglets up to 6 weeks of age.

Immune response does not prevent homologous Porcine epidemic diarrhoea virus reinfection five months after the initial challenge.

The aim of the present study was to evaluate the duration of protective immunity against Porcine epidemic diarrhoea virus (PEDV). To do so, a two phases study was performed. In the first phase, 75 four-week-old pigs (group A) were orally inoculated (0 days post-inoculation; dpi) with a European PEDV G1b strain and 14 were kept as controls (group B). The second phase started five months later (154 dpi), when animals in group A were homologous challenged and animals in group B were challenged for first time. Clinical signs, viral shedding and immune responses were evaluated after each inoculation, including the determination of antibodies (ELISA and viral neutralization test, IgA and IgG ELISPOTs using peripheral blood mononuclear cells and lymph node cells) and the frequency of interferon-gamma (IFN-γ) secreting cells. During the first phase, loose stools/liquid faeces were observed in all group A animals. Faecal shedding of PEDV occurred mostly during the first 14 days but, in some animals, persisted until 42 dpi. All inoculated animals seroconverted for specific-PEDV IgG and IgA, and for neutralizing antibodies (NA). At 154 dpi, 77% of pigs were still positive for NA. After that, the homologous challenge resulted in a booster for IgG, IgA, NA, as well as specific-PEDV IgG, IgA and IFN-γ secreting cells. In spite of that, PEDV was detected in faeces of all pigs from group A, indicating that the immune response did not prevent reinfection, although the duration of the viral shedding and the total load of virus shed were significantly lower for previously challenged pigs (p < .05). Taken together, the results indicated that, potentially, maintenance of PEDV infection within an endemic farm may occur by transmission to and from previously infected animals and also indicates that sterilizing immunity is shorter than the productive life of pigs.

First identification and characterization of rotavirus H in swine in Spain.

Rotaviruses (RVs) are classified into nine species or groups (RVA-RVD and RVF-RVJ). RVA, RVB and RVC are well-recognized as etiological agents of enteric disease on swine farms and have been identified in all countries with a relevant pork production. Contrarily, RVH has only been identified on swine farms from Japan and more recently from Brazil, USA, South Africa and Vietnam but not yet in Europe. The occurrence of RVH was investigated in 103 Spanish pig herds. Nine farms were positive and we achieved a complete nucleotide sequences in three isolates while another RVH isolate was partially sequenced. Mean nucleotide identities with the RVH sequences available in GenBank ranged between 69.4% and 93.7%. Phylogenetically, all genomic segments of Spanish RVH isolate clustered closely with other porcine RVH strains but were distantly related to human RVH as well as bat RVH strain. To the best of our knowledge, this is the first report of RVH on swine farms in Europe including its characterization by means of complete genome sequencing.

Phylogenetic relationships investigation of Mycobacterium caprae strains from sympatric wild boar and goats based on whole genome sequencing.

Tuberculosis (TB) in wildlife challenges epidemiological surveillance and disease control. An outbreak of TB was detected in a free-ranging wild boar population of a Natural Park in Catalonia (Spain) and the outbreak investigation was conducted in the area. During the study period (2015-2020), 278 wild boars were analysed by gross pathology, histopathology, mycobacterial culture and DVR-spoligotyping. In addition, all cattle (49) and goat (47) herds of the area were tested with tuberculin skin test. TB compatible lesions were detected in 21 wild boars, and Mycobacterium caprae was isolated in 17 of them with two different spoligotypes: SB0415 (13) and SB1908 (4). Only two goat herds showed TB positive animals that were subsequently slaughtered. M. caprae with the spoligotypes SB0416 and SB0415 were isolated from these animals. To investigate the phylogenetic relationships and the transmission chain of the outbreak, nine strains isolated from six wild boars and three goats of the study area were analysed by whole genome sequencing (WGS) followed by single nucleotide polymorphism (SNP) analysis by maximum likelihood and median-joining network inference methods. Results indicated that infected wild boars maintained M. caprae strains circulation in their own population and have likely transmitted the infection to goats, thus acting as TB reservoirs, compromising the success of livestock TB eradication campaigns and posing a risk for public health. The results also highlighted the usefulness of WGS followed by SNP analysis in providing relevant epidemiological information when detailed contact data are missing.

Dual Host and Pathogen RNA-Seq Analysis Unravels Chicken Genes Potentially Involved in Resistance to Highly Pathogenic Avian Influenza Virus Infection.

Highly pathogenic avian influenza viruses (HPAIVs) cause severe systemic disease and high mortality rates in chickens, leading to a huge economic impact in the poultry sector. However, some chickens are resistant to the disease. This study aimed at evaluating the mechanisms behind HPAIV disease resistance. Chickens of different breeds were challenged with H7N1 HPAIV or clade 2.3.4.4b H5N8 HPAIV, euthanized at 3 days post-inoculation (dpi), and classified as resistant or susceptible depending on the following criteria: chickens that presented i) clinical signs, ii) histopathological lesions, and iii) presence of HPAIV antigen in tissues were classified as susceptible, while chickens lacking all these criteria were classified as resistant. Once classified, we performed RNA-Seq from lung and spleen samples in order to compare the transcriptomic signatures between resistant and susceptible chickens. We identified minor transcriptomic changes in resistant chickens in contrast with huge alterations observed in susceptible chickens. Interestingly, six differentially expressed genes were downregulated in resistant birds and upregulated in susceptible birds. Some of these genes belong to the NF-kappa B and/or mitogen-activated protein kinase signaling pathways. Among these six genes, the serine protease-encoding gene PLAU was of particular interest, being the most significantly downregulated gene in resistant chickens. Expression levels of this protease were further validated by RT-qPCR in a larger number of experimentally infected chickens. Furthermore, HPAIV quasi-species populations were constructed using 3 dpi oral swabs. No substantial changes were found in the viral segments that interact with the innate immune response and with the host cell receptors, reinforcing the role of the immune system of the host in the clinical outcome. Altogether, our results suggest that an early inactivation of important host genes could prevent an exaggerated immune response and/or viral replication, conferring resistance to HPAIV in chickens.

Diversity of influenza A viruses retrieved from respiratory disease outbreaks and subclinically infected herds in Spain (2017-2019).

The present study was aimed to assess the diversity of influenza A viruses (IAV) circulating in pig farms in the Iberian Peninsula. The study included two different situations: farms suffering respiratory disease outbreaks compatible with IAV (n = 211) and randomly selected farms without overt respiratory disease (n = 19). Initially, the presence of IAV and lineage determination was assessed by qRT-PCR using nasal swabs. IAV was confirmed in 145 outbreaks (68.7%), mostly in nurseries (53/145; 36.5%). Subtyping by RT-qPCR was possible in 94 of those cases being H1avN2hu (33.6%), H1avN1av (24.3%) and H1huN2hu (18.7%), the most common lineages. H3huN2hu and H1pdmN1pdm represented 7.5% and 6.5% of the cases, respectively. As for the randomly selected farms, 15/19 (78.9%) were positive for IAV. Again, the virus was mostly found in nurseries and H1avN2hu was the predominant lineage. Virus isolation in MDCK cells was attempted from positive cases. Sixty of the isolates were fully sequenced with Illumina MiSeq®. Within those 60 isolates, the most frequent genotypes had internal genes of avian origin, and these were D (19/60; 31.7%) and A (11/60; 18.3%), H1avN2hu and H1avN1av, respectively. In addition, seven previously unreported genotypes were identified. In two samples, more than one H or N were found and it was not possible to precisely establish their genotypes. A great diversity was observed in the phylogenetic analysis. Notably, four H3 sequences clustered with human isolates from 2004-05 (Malaysia and Denmark) that were considered uncommon in pigs. Overall, this study indicates that IAV is a very common agent in respiratory disease outbreaks in Spanish pig farms. The genetic diversity of this virus is continuously expanding with clear changes in the predominant subtypes and lineages in relatively short periods of time. The current genotyping scheme has to be enlarged to include the new genotypes that could be found in the future.

The use of H-index to assess research priorities in poultry diseases.

Identifying which diseases represent a priority is crucial to optimize resources for diagnostics, control, and prevention. Here, the impact of 111 poultry pathogens belonging to Viruses (n = 31), Bacteria (n = 33), and Other (n = 47) was assessed using the H-index. The overall mean H-indexes suggested that poultry Viruses have statistically greater impact than Bacteria, which in turn are statistically more relevant than Others. Among the 20 highest H-indexes, 45% were zoonotic, and almost a third was Office International des Epizooties-listed. Avian influenza virus (H-index 127), Salmonella enteritidis and Salmonella typhimurium (H-index 72), and Eimeria spp (H-index 70) ranked the highest in Virus, Bacteria, and Other, respectively. Pathogens that produce overt clinical diseases and economic damage, cause immunosuppression, and/or are zoonotic had the highest H-index scores. The evolution of citations of particular pathogens reflected severe poultry outbreaks and/or zoonotic outbreaks in relatively wide geographic areas. Also, the evolution of citations based on taxonomic groups mirrored major changes in poultry production practices and management throughout history. Thus, Others were the most cited pathogens until the 1970s and, following 3 decades of unpopularity because of widespread use of intensive production practices, regained importance in the 2000s thanks to welfare regulation changes. Citations for Bacteria increased especially from the 1990s onward, probably because of the ban of growth promoters in western countries and the need to find new control methods for bacterial and protozoal infections. In general, countries with the greatest poultry production and research budgets had higher research production, that is the United States of America (USA) and China. Interestingly, the United Kingdom was among the top research producers despite falling behind other countries in poultry production and research budget. Moreover, the USA exhibited the strongest poultry research production based on number and diversity of publications (Dcos-index). In conclusion, the H-index could be a valid, simple tool to prioritize funding or interest in poultry diseases, especially when used as a preliminary selection approach in combination with other metrics.