Whole-Genome Sequencing of Two Canine Herpesvirus 1 (CaHV-1) Isolates and Clinicopathological Outcomes of Infection in French Bulldog Puppies.

Canine herpesvirus 1 (CaHV-1) infects dogs, causing neonatal death and ocular, neurological, respiratory, and reproductive problems in adults. Although CaHV-1 is widespread in canine populations, only four studies have focused on the CaHV-1 whole genome. In such context, two CaHV-1 strains from both the kidney and spleen of 20-day-old deceased French Bulldog puppies were recently isolated in Sardinia, Italy. The extracted viral DNA underwent whole-genome sequencing using the Illumina MiSeq platform. The Italian CaHV-1 genomes were nearly identical (>99%), shared the same tree branch, and clustered near the ELAL-1 (MW353125) and BTU-1 (KX828242) strains, enlarging the completely separated clade discussed by Lewin et al., in 2020. This study aims to provide new insights on the evolution of the CaHV-1, based on high-resolution whole-genome phylogenetic analysis, and on its clinicopathological characterization during a fatal outbreak in puppies.

The Global Evolutionary History of Orf Virus in Sheep and Goats Revealed by Whole Genomes Data.

Orf virus (ORFV) belongs to the genus Parapoxvirus (Poxviridae family). It is the causative agent of contagious ecthyma (CE) that is an economically detrimental disease affecting small ruminants globally. Contagious ecthyma outbreaks are usually reported in intensive breeding of sheep and goats but they have also been reported in wildlife species. Notably, ORFV can infect humans, leading to a zoonotic disease. This study aims to elucidate the global evolutionary history of ORFV genomes in sheep and goats, including the first genomes from Central America in the analyses. In comparison to the last study on ORFV whole genomes, the database now includes 11 more sheep and goat genomes, representing an increase of 42%. The analysis of such a broader database made it possible to obtain a fine molecular dating of the coalescent time for ORFV S and G genomes, further highlighting the genetic structuring between sheep and goat genomes and corroborating their emergence in the latter half of 20th century.

Origin, Genetic Variation and Molecular Epidemiology of SARS-CoV-2 Strains Circulating in Sardinia (Italy) during the First and Second COVID-19 Epidemic Waves.

Understanding how geography and human mobility shape the patterns and spread of infectious diseases such as COVID-19 is key to control future epidemics. An interesting example is provided by the second wave of the COVID-19 epidemic in Europe, which was facilitated by the intense movement of tourists around the Mediterranean coast in summer 2020. The Italian island of Sardinia is a major tourist destination and is widely believed to be the origin of the second Italian wave. In this study, we characterize the genetic variation among SARS-CoV-2 strains circulating in northern Sardinia during the first and second Italian waves using both Illumina and Oxford Nanopore Technologies Next Generation Sequencing methods. Most viruses were placed into a single clade, implying that despite substantial virus inflow, most outbreaks did not spread widely. The second epidemic wave on the island was actually driven by local transmission of a single B.1.177 subclade. Phylogeographic analyses further suggest that those viral strains circulating on the island were not a relevant source for the second epidemic wave in Italy. This result, however, does not rule out the possibility of intense mixing and transmission of the virus among tourists as a major contributor to the second Italian wave.

Experimental infection of Aedes (Stegomyia) albopictus and Culex pipiens mosquitoes with Bluetongue virus.

Bluetongue disease (BT), caused by Bluetongue virus (BTV), infects wild and domestic ruminants, causing severe economic damage in the cattle and sheep industry. Proven vectors of BTV are biting midges belonging to the Culicoides genus, but other arthropods are considered potential vectors, such as ticks, mosquitoes, wingless flies, and sand flies. The present study represents the first attempt to evaluate the vectorial capacity of Culex pipiens and Aedes albopictus for BTV. Mosquitoes were artificially fed with blood containing BTV serotype 1. Infection, dissemination and transmission rates were evaluated at 0, 3, 7, 14 and 21 days after an infected blood meal. Viral RNA was only detected up to 3 days post infection in the bodies of both species. This study indicates that the two Italian populations of Cx. pipiens and Ae. albopictus are not susceptible to BTV infection.

Malignant Catarrhal Fever in Sardinia (Italy): A Case Report.

Using a multidisciplinary approach, this report describes a clinical case of malignant catarrhal fever (MCF) occurring in a calf, which shared the pasture with sheep on a farm located in the island of Sardinia (Italy). We confirmed the conventional clinico-histopathological features of MCF, as well was the presence of Ovine herpesvirus type 2 (OvHV-2) DNA in several tissues, employing histological and virological investigations. The phylogenetic analysis revealed that this Sardinian OvHV-2 strain is genetically similar to all the other Italian strains. By Real Time PCR examinations of blood samples collected across Sardinia's sheep population, which is considered the most important reservoir species, we discovered an OvHV-2 prevalence ranging from 20 to 30 percent. Despite the high prevalence of OvHV-2 in the Sardinian sheep population, clinical disease in bovine remains sporadic; further investigations are needed to understand the risk factors that regulate this epidemiological aspect.

Entomological surveillance of Zika virus in Sardinia, Italy, 2016.

Zika Virus (ZIKV) is a RNA virus belonging to the genus Flavivirus, family Flaviviridae. This virus is transmitted through bite of Aedes mosquitoes, in particular Ae. aegypti. On February 1st 2016, the World Health Organization (WHO) has declared ZIKV a Public Health Emergency of International Concern. Successively, considering the establishment of Ae. albopictus, WHO has classified Italy as having a moderate likelihood of local transmission of ZIKV, preceded in Europe only by France. For this reason an entomological surveillance plan was been activated in Sardinia in 2016. BG Sentinel Mosquito Traps have been positioned in 29 sites, comprising urban areas and points of entry, as ports and airports. Mosquitoes were collected fortnightly from April to December. A total of 3,089 mosquitoes were collected belonging to 10 species. The most numerous species have been Cx. pipiens s.l. and Ae. albopictus. All mosquitoes sampled have been assayed by real time reverse transcriptase PCR for detection of ZIKV RNA. A total of 584 pool have been analyzed and have been reported no evidence of ZIKV. A permanent entomological surveillance should be implemented principally in the urban areas and points of entry, as ports and airports, because Ae. albopictus, susceptible to ZIKV, is established in Sardinia and also know the recent introduction of invasive mosquitoes species Ae. koericus and Ae. japonicus in Italy.

Testicular Degeneration and Infertility following Arbovirus Infection.

Arboviruses can cause a variety of clinical signs, including febrile illness, arthritis, encephalitis, and hemorrhagic fever. The recent Zika epidemic highlighted the possibility that arboviruses may also negatively affect the male reproductive tract. In this study, we focused on bluetongue virus (BTV), the causative agent of bluetongue and one of the major arboviruses of ruminants. We show that rams that recovered from bluetongue displayed signs of testicular degeneration and azoospermia up to 100 days after the initial infection. Importantly, testicular degeneration was induced in rams experimentally infected with either a high (BTV-1IT2006)- or a low (BTV-1IT2013)-virulence strain of BTV. Rams infected with the low-virulence BTV strain displayed testicular lesions in the absence of other major clinical signs. Testicular lesions in BTV-infected rams were due to viral replication in the endothelial cells of the peritubular areas of the testes, resulting in stimulation of a type I interferon response, reduction of testosterone biosynthesis by Leydig cells and destruction of Sertoli cells and the blood-testis barrier in more severe cases. Hence, BTV induces testicular degeneration and disruption of spermatogenesis by replicating solely in the endothelial cells of the peritubular areas unlike other gonadotropic viruses. This study shows that a naturally occurring arboviral disease can cause testicular degeneration and affect male fertility at least temporarily.IMPORTANCE During the recent Zika epidemic, it has become apparent that arboviruses could potentially cause reproductive health problems in male patients. Little is known regarding the effects that arboviruses have on the male reproductive tract. Here, we studied bluetongue virus (BTV), an arbovirus of ruminants, and its effects on the testes of rams. We show that BTV was able to induce testicular degeneration in naturally and experimentally infected rams. Testicular degeneration was caused by BTV replication in the endothelial cells of the peritubular area surrounding the seminiferous tubules (the functional unit of the testes) and was associated with a localized type I interferon response, destruction of the cells supporting the developing germinal cells (Sertoli cells), and reduction of testosterone synthesis. As a result of BTV infection, rams became azoospermic. This study highlights that problems in the male reproductive tract caused by arboviruses could be more common than previously thought.

Persistence of Bluetongue virus serotype 1 virulence in sheep blood refrigerated for 10 years.

This paper reports that Bluetongue virus serotype 1 (BTV-1) infected blood collected during the 2006 Sardinia (Italy) epidemic from a ewe with clinical disease and stored at ~ 5°C for 10 years, caused Bluetongue (BT)-like clinical disease and death when inoculated into a susceptible Sarda breed ram. Anatomo-histopathological examination and Real-Time Reverse Transcriptase PCR (Real-Time RT-PCR) confirmed the presence of BTV-1 in several tissues proving that the BTV-1 2006 isolate has maintained its infectivity and virulence.

Novel putative Bluetongue virus in healthy goats from Sardinia, Italy.

In recent years, novel Bluetongue virus (BTV) serotypes have been isolated and/or sequenced by researchers within the field. During Bluetongue surveillance activities, we identified a putative novel BTV serotype in healthy goats from Sardinia, Italy. RNAs purified from blood and serum samples were positive for BTV by a generic real time RT-PCR and c-ELISA, respectively, whereas genotyping and serotyping were unsuccessful. By NGS, the whole genome sequence was obtained from two blood samples (BTV-X ITL2015 strains 34200 and 33531). Overall, Seg 2 of BTV-X ITL2015 shows the highest identity (75.3-75.5% nt/77.4-78.1% aa) with recently isolated BTV-27s from Corsica and with the last discovered BTV XJ1407 from China (75.9% nt /78.2% aa), whereas it is less related with BTV-25 from Switzerland (73.0% nt/75.0% aa) and BTV-26 from Kuwait (62.0% nt/60.5% aa). A specific RT-qPCR targeting Seg 2 of BTV-X ITL2015 was assessed in this study. Considering the Seg 2/VP2 identity of BTV-X ITL2015 with BTV-25, 26, 27s and BTV XJ1407 and that serum of BTV-X ITL2015 infected goats failed to neutralize all tested extant serotypes, we propose the existence of a novel BTV serotype circulating in goats in Sardinia. Isolation was so far unsuccessful thus hampering proper antigenic characterization.

Bluetongue virus serotypes 1 and 4 in Sardinia during autumn 2012: new incursions or re-infection with old strains?

Since 2000 several bluetongue virus (BTV) incursions have occurred in Sardinia (Italy) involving serotypes 1, 2, 4, 8 and 16. In October 2012, new BT outbreaks caused by BTV-1 and BTV-4 were reported. Nearly 500 flocks were infected and 9238 sheep died because of the infection. When sequenced, Seg-10 of both strains shared 99% similarity at nucleotide level with BTV strains that have circulated in the Mediterranean basin in the last few years. Similarly, Seg-5 sequences of BTV-1 and BTV-4 newly isolated Sardinian strains are identical and cluster together with recent BTV-1 circulating in the Mediterranean Basin and the BTV-4 strains isolated in Tunisia in 2007 and 2009. These BTV-4 strains differ from the ones that circulated in Europe from 2003 to 2005 and appear to be reassortant strains.

Growth hormone gene variability and its effects on milk traits in primiparous Sarda goats.

The aim of this research was to investigate variability in each of the five exons of the caprine growth hormone (gGH) gene, in order to establish the possible relationships with milk traits in Sarda breed goat. The general linear model procedure was used to analyse the effects of the single strand conformation (SSCP) profiles on milk traits of 100 lactating goats. Analysis of conformational polymorphism at exons 1-5 revealed a total of 25 differing banding patterns. Sequencing revealed 21 nucleotide changes (compared with GenBank D00476): 14 were polymorphic and 7 monomorphic; 19 in exonic regions, 5 of which were nonsynonymous. A SNP upstream of the transcription initiation codon (c.-3A>G) and an indel (c.*29_30insC) in the 3'UTR, were detected. Alignment of 4 cloned sequences including the entire gGH gene led to the identification of 22 nucleotide variations within the intron regions, including two indels. Association analysis revealed that each exon, except exon-1, affected milk yield, exons 1 and 3 influenced milk fat percentage, and all exons, except exon-2, had an effect on protein percentage, supporting previous results in livestock. The variability detected at the caprine GH gene might provide useful information for the phylogeny of ruminants and, more importantly, have implications on the biological function of the growth hormone and on those traits resulting from its physiological action, including milk production and composition. The caprine GH gene may become a useful molecular marker for a more effective genetic selection for milk production traits in goats.