Identification and genomic characterization of influenza viruses with different origin in Mexican pigs

Swine influenza is a worldwide disease, which causes damage to the respiratory system of pigs. The H1N1 and H3N2 subtypes circulate mainly in the swine population of Mexico. There is evidence that new subtypes of influenza virus have evolved genetically and have been rearranged with human viruses and from other species; therefore, the aim of our study was to identify and characterize the genetic changes that have been generated in the different subtypes of the swine influenza virus in Mexican pigs. By sequencing and analyzing phylogenetically the eight segments that form the virus genome, the following subtypes were identified: H1N1, H3N2, H1N2 and H5N2; of which, a H1N1 subtype had a high genetic relationship with the human influenza virus. In addition, a H1N2 subtype related to the porcine H1N2 virus reported in the United States was identified, as well as, two other viruses of avian origin from the H5N2 subtype. Particularly for the H5N2 subtype, this is the first time that its presence has been reported in Mexican pigs. The analysis of these sequences demonstrates that in the swine population of Mexico, circulate viruses that have suffered punctual‐specific mutations and rearrangements of their proteins with different subtypes, which have successfully adapted to the Mexican swine population.     

First evidence of bovine immunodeficiency virus infection in Mexican cattle

This study set out to identify the presence of bovine immunodeficiency virus (BIV) in animals geographically located in Mexico. BIV was first discovered in the United States in a dairy cow with persistent lymphocytosis, lymphoid hyperplasia and lymphocytic encephalitis. Many studies indicate that BIV infection is globally distributed, but its presence in Mexico remains unknown. We collected 1,168 heparinized blood samples from cattle in ten states across the Mexican Republic, then separated plasma using centrifugation and tested for antibodies against BIV. We used an indirect ELISA based on the use of a synthetic peptide derived from transmembrane glycoprotein (gp45/TM). In order to identify the viral genome, we designed a synthetic gene as a PCR control, as well as a pair of oligonucleotides for amplifying a 519 bp product of the env gene which encodes the surface protein. Positive amplicons were purified and subjected to nucleotide sequencing. A total of 189 (28.94%) tested plasma samples suggest the presence of specific anti‐BIV antibodies in all states studied except for Chiapas. Additionally, PCR results identified six positive cows in the states of Puebla and Coahuila. BIV in these cows was confirmed via nucleotide sequencing and in silico analysis of these samples. This is the first report of the presence of BIV in Mexican cattle.     

Identification and characterization of lumpy skin disease virus isolated from cattle in the Republic of North Ossetia‐Alania in 2015

The first notifications of the unknown disease of cattle appeared in September–October 2015 in North Caucasus region of Russia (Republic of North Ossetia‐Alania). The clinical signs included watery discharge from eyes, apathy, loss of appetite, salivation, lameness and nodular skin lesions. Capripoxvirus genome was detected by real‐time PCR in the tissue samples of sick animals. The aetiological agent was isolated in the primary cell cultures of lamb testis and goat testis, as well as in the continuous MDBK cell culture. Further sequencing of the GPCR gene and phylogenetic analysis showed the close genetic relationship of isolated capripoxvirus with a group of lumpy skin disease virus. Koch's postulates were fulfilled by the experimental infection of four calves with a suspension of tissue samples from sick animals.     

Emergence and expansion of novel pathogenic reassortant strains of infectious bursal disease virus causing acute outbreaks of the disease in Europe

Infectious bursal disease virus (IBDV) is the aetiological agent of a highly contagious chicken immunodeficiency disorder known as Gumboro disease, which cause severe economic loses to the poultry worldwide. The emergence of very virulent IBDV strains (vvIBDV) during the late 80s resulted in drastic changes to the epidemiology of IBDV with a dramatic increase in the mortality of the animals affected. Molecular studies determined that the emergence of the vvIBDV was a consequence of a genomic reorganization of IBDV known as reassortant event by which the virus combined two emergent genetic background vvIBDV for segment A and vvIBDV for segment B. In the current study, a retrospective analysis was conducted, and samples collected during acute outbreaks of Gumboro disease in Poland during 1992–2015 were submitted to sequencing and further molecular and phylogenetic analyses. The results obtained not only revealed a high genetic diversity for Polish IBDV strains but a new population of IBDV was identified. These novel reassortant strains with a unique genetic background contain the segment A from very virulent strains and segment B from an unidentified source, phylogenetically segregated and classified as ‘transitional lineage’. The results obtained also showed the presence of this new lineage in Finland, evidencing the expansion of this new genomic reorganized viral strain in Europe representing an additional threat to the global situation of IBDV.     

Shuni virus in Israel: Neurological disease and fatalities in cattle

The insect‐transmitted Shuni virus (SHUV) belongs to the Simbu serogroup of orthobunyaviruses and it is known to induce abortions, stillbirths and severe congenital malformations in ruminants and may cause neurological signs in infected horses. Here, SHUV was detected in brain samples of two Israeli cattle, which suffered from severe neurological signs that led to the deaths of the animals. During histopathological examination of the first case, a 5‐month‐old calf, small perivascular cuffs, composed mainly of neutrophils with few lymphocytes were observed in the brain stem and cerebrum. Similar infiltrates were also found to a lesser extent in the cerebellar meninges leading to the diagnosis of acute‐subacute meningoencephalitis. The histological examination of the brainstem from the second case, a 16‐month‐old heifer, revealed perivascular infiltration composed of equal numbers of macrophages and neutrophils associated with cerebral and meningeal haemorrhages. In this case encephalitis was diagnosed. Viral RNA was extracted from brain samples of both cattle that suffered from severe neurological signs and was subsequently tested by a polymerase chain reaction PCR assay specific for Simbu serogroup viruses and found positive. The presence of SHUV was subsequently confirmed by the isolation of the virus from one sample and sequence analysis of both brain samples. The comparison of the complete sequences of the coding regions of all three genome segments from both cases revealed a close relationship to Shuni viruses detected in tissue samples of aborted or malformed calves or lambs born during the last years in Israel.     

Epidemiological investigation of H9 avian influenza virus,Newcastle disease virus,Tembusu virus,goose parvovirus and goose circovirus infection of geese in China

To deepen the knowledge about epidemic prevalence in the goose breeding field, a triplex PCR assay was established, and 478 samples were collected from scaled goose farms in 11 provinces in China. The results of this epidemiological investigation showed that incidence rates of H9 avian influenza and goose circovirus were the highest among five infectious diseases that were evaluated. In addition, the triplex PCR assay established remarkable sensitivity, rapidity and versatility compared to other diagnostic methods. Dual infection comprised a large proportion of the co‐infections in the field, of which the combinations of H9/Tembusu, H9/goose circovirus and goose circovirus/Tembusu co‐infected cases were more common. Epidemics were more severe in winter and spring. Additionally, significant differences in the prevalence of these infectious diseases were observed in association with different age groups. In addition, phylogenetic analysis, determined by the neighbour‐joining method, was carried out to investigate the evolution of these viruses during the study period. For the most part, virus strains isolated during the study were consistent with most goose‐origin strains isolated from the Chinese mainland over the past few years. However, mutations were observed between isolated H9 avian influenza virus strains and sequences available from GenBank, which should draw much attention.     

Experimental infection with high‐ and low‐virulence strains of border disease virus (BDV) in Pyrenean chamois (Rupicapra p. pyrenaica) sheds light on the epidemiological diversity of the disease

Since 2001, Pyrenean chamois (Rupicapra pyrenaica pyrenaica) populations have been affected by border disease virus (BDV) causing mortalities of more than 80% in some areas. Field studies carried out in France, Andorra, and Spain have shown different epidemiological scenarios in chamois populations. This study was designed to confirm the presence of BDV strains of a high and low virulence in free‐ranging chamois populations from Pyrenees and to understand the implications of these findings to the diverse epidemiological scenarios. An experimental infection of Pyrenean chamois with a high‐virulence (Cadí‐6) and low‐virulence (Freser‐5) BDV strains was performed. Pregnant and non‐pregnant animals with and without antibodies against BDV were included in each group. Cadí‐6 BDV strain was confirmed to be of high virulence for seronegative adults and their foetuses. The antibody negative chamois infected with Freser‐5 BDV strain did not show symptoms, presented less viral distribution and RNA load in tissues than Cadí‐6 group, and cleared the virus from the serum. However, foetuses died before the end of the experiment and RNA virus was detected in sera and tissues although with lower RNA load than the Cadí‐6 group. Chamois from both groups presented lesions in brain but the ones infected with the low‐virulence Freser‐5 BDV strain were mild and most likely transient. In both groups, seropositive pregnant females and all but one of their foetuses did not present viraemia or viral RNA in tissues. The existence of a low‐virulence strain has been confirmed experimentally and related to chamois population infection dynamics in the area where it was isolated. Such strain may persist in the chamois population through PI animals and may induce cross‐protection in chamois against high‐virulence strains. This study demonstrates that viral strain diversity is a significant factor in the heterogeneity of epidemiological scenarios in Pyrenean chamois populations.     

Detection of vaccine‐like lumpy skin disease virus in cattle and Musca domestica L. flies in an outbreak of lumpy skin disease in Russia in 2017

Since 2012, lumpy skin disease virus (LSDV) has been spreading from the Middle East to south‐east Europe and Russia. Although vaccination campaigns have managed to contain LSDV outbreaks, the risk of further spread is still high. The most likely route of LSDV transmission in short distance spread is vector‐borne. Several arthropod species have been suggested as potential vectors, but no proven vector has yet been identified. To check whether promiscuous‐landing synanthropic flies such as the common housefly (Musca domestica ) could be involved, we carried out entomological trapping at the site of a recent LSDV outbreak caused by a vaccine‐like LSDV strain. The presence of vaccine‐like LSDV DNA was confirmed by the assay developed herein, the assay by Agianniotaki et al. (2017) and RPO 30 gene sequencing. No evidence of field LSDV strain circulation was revealed. In this study, we discovered that M. domestica flies carried vaccine‐like LSDV DNA (C _t  > 25.5), whereas trapped stable flies from the same collection were negative for both field and vaccine LSDV . To check whether flies were contaminated internally and externally, 50 randomly selected flies from the same collection were washed four times and tested. Viral DNA was mainly detected in the 1st wash fluid, suggesting genome or even viral contamination on the insect cadaver. In this study, internal contamination in the insect bodies without differentiation between the body locations was also revealed; however, the clinical relevance for mechanical transmission is unknown. Further work is needed to clarify a role of M. domestica in the transmission of LSDV . To our knowledge, this is the first report demonstrating that an attenuated LSD vaccine strain has been identified in Russian cattle given the ban on the use of live attenuated vaccines against LSDV.     

Detection of influenza D virus in bovine respiratory disease samples,UK

Influenza D is a newly described virus of cattle, pigs and small ruminants first detected in North America during 2011. Cattle have been shown to be the main viral reservoir and mounting evidence indicates that infection with influenza D may contribute to the development of bovine respiratory disease. The virus has been detected across the United States, Europe and Asia. To date, influenza D has not been reported in the UK. During the winter and spring of 2017/2018, we performed molecular testing of cattle submitted for post‐mortem examination where respiratory disease signs were present. We detected influenza D virus in 8.7% of cases, often as the sole viral agent and always in conjunction with bacterial co‐infection with one or more agents. Viral RNA was present in both the upper and lower respiratory tract and pathological changes in lung tissues were observed alongside signs of concurrent bacterial infections. Sequencing of one UK isolate revealed that it is similar to viruses from the Republic of Ireland and Italy.     

Detection of porcine circovirus‐like virus P1 in Hebei,China

Porcine circovirus‐like virus P1 is a novel unclassified circovirus that was first detected in China and may be associated with post‐weaning multisystemic wasting syndrome (PMWS ) and congenital tremor. In this study, we detected P1 infection in pigs in Hebei Province, China, in 2017. One hundred and forty of 500 (28.0%) serum samples from 25 pig farms with different PMWS status in seven cities were P1 positive on PCR . Twelve P1 strains were sequenced, and the complete genomes of 11 P1 strains were 648 nucleotides (nt) in length, whereas that of strain ZJK 02 was 647 nt, with a G deletion at position of 183 in its genome. The complete genomic and capsid protein sequences of the 12 P1 strains analysed in this study shared 98.8%–100.0% and 86.5%–100.0% identity, respectively. A phylogenetic analysis based on the complete genomic and capsid sequences of 26 P1 strains showed that the 12 P1 sequences from Hebei Province clustered on two small branches. Further studies of the evolution and pathogenesis of P1 are required.     

Emergence of novel Seneca Valley virus strains in China, 2017

Since early 2017, several outbreaks of Seneca Valley virus (SVV) infection have re‐emerged in China. We report the identification of novel SVV KS15‐01‐like strains showing a notable distinction with previous Chinese SVV strains. The determined SVV strains are currently causing new outbreaks in two provinces in China where no SVV infection has been reported previously, implying the increased epidemic regions and potential threat to local pig breeds of SVV in China.     

Foot‐and‐mouth disease virus serotype SAT1 in cattle,Nigeria

The knowledge of foot‐and‐mouth disease virus (FMDV) dynamics and epidemiology in Nigeria and the West Africa subregion is important to support local and regional control plans and international risk assessment. Foot‐and‐mouth disease virus serotype South African territories (SAT)1 was isolated, identified and characterized from an FMD outbreak in cattle in Nigeria in 2015, 35 years after the last report of FMDV SAT1 in West Africa. The VP1 coding sequence of the Nigerian 2015 SAT1 isolates diverges from reported SAT1 topotypes resulting in a separate topotype. The reporting of a novel FMDV SAT1 strain in the virus pool 5 (West and Central Africa) highlights the dynamic and complex nature of FMDV in this region of Africa. Sustained surveillance is needed to understand the origin, the extent and distribution of this novel SAT1 topotype in the region as well as to detect and monitor the occurrence of (re‐)emerging FMDV strains.     

Re‐emergence of a genotype VIII virulent Newcastle disease virus isolated from Chinese game fowl after 13 years

Circulation of dominant genotypes VI and VII of Newcastle disease virus (NDV) is causing significant economic losses to the poultry industry in China. However, reports of Newcastle disease (ND) outbreaks caused by genotype VIII strains of NDV are rare. In this study, a virulent genotype VIII strain of NDV, designated GXGB2011, was isolated from a vaccinated game fowl flock showing clinic signs of infection in Pinxiang county, Guangxi, China. The whole genome of the isolate was completely sequenced and was found to be comprised of 15,192 nucleotides (nt), encoding the six structural proteins in the order of 3′‐NP‐P‐M‐F‐HN‐L‐5′. The pattern of cleavage site ¹¹²RRQKR↓F¹¹⁷ in the fusion (F) protein and the intracerebral pathogenicity index (ICPI) value of 1.5 showed that the strain GXGB2011 was a velogenic NDV. The results of the challenge experiment with the 5‐week‐old SPF chickens showed that the strain was highly pathogenic with 100% morbidity and mortality of the challenged birds. Based on the detection of virus in different organs of the infected birds, the highest viral load in caecal tonsils was observed and viral levels in immune organs were higher than those in the respiratory organs. Bayesian reconstruction of complete genomes based on the sequences of 66 NDV reference strains showed that the strain belonged to the genotype VIII of NDV. Phylogenetic analysis showed that the strain was more closely related to the foreign strains gamefowl/U.S.(CA)/24225/98, 1ITTY94060 and IT‐147/94 rather than to the first domestic strains of the emergence genotype VIII in Qinghai, China during 1979–1985. In summary, the results of the study demonstrated the re‐emergence of a highly pathogenic virulent isolate of genotype VIII of NDV. These results indicate the risk that this genotype VIII of NDV may spread to commercial chickens from game fowl.     

Molecular detection of hepatitis E virus in wild boar population in eastern Romania

In industrialized countries, Hepatitis E is a recognized zoonosis, with wild boar and swine representing the main reservoirs for zoonotic genotype HEV ‐3 in Europe. Data related to HEV infection in wild boar population in Romania are restricted to serological surveys. Therefore, our main goal was to determine the HEV prevalence in wild boar population and to characterize HEV strains circulating in Romania. Using TaqMan real‐time RT ‐PCR assay, we analyzed the presence of RNA HEV in 45 liver samples and five spleen samples collected from 50 wild boars. Samples were collected during the 2013–2015 hunting seasons. Nine samples of 50 were tested positive for HEV RNA , resulting an overall prevalence of 18%. Phylogenetic analysis revealed that the isolates clustered in different HEV ‐3 monophyletic groups, depending on the sampling county. This is the first study signalling, based on molecular analysis, the presence of HEV in wild boar population from Romania. Also, in this study, we report the detection of HEV in splenic tissue from wild boar.     

Novel serotype of bluetongue virus in South America and first report of epizootic haemorrhagic disease virus in Ecuador

Bluetongue virus (BTV ) and Epizootic haemorrhagic disease virus (EHDV ) are closely related Orbiviruses that affect domestic and wild ruminants. In Ecuador previous serological studies reported the presence of BTV ; however, no data are available about the presence of EHDV . In this study, 295 cattle without symptoms of infection were sampled from two farms located in Andean and Amazonian regions and from a slaughterhouse in the coastal region. ELISA analyses showed high prevalence of BTV (98.9%) and EHDV (81.3%) antibodies, and RT ‐qPCR s revealed the presence of EHDV (24.1%) and BTV (10.2%) genomes in cattle blood samples. Viral isolation allowed to identify EHDV serotype 1 (EHDV 1) and BTV serotypes 9 (BTV 9), 13 and 18. These findings suggest that BTV and EHDV are enzootic diseases in Ecuador.     

Evidence for Tunisian‐Like Pestiviruses Presence in Small Ruminants in Italy Since 2007

The genus Pestivirus, which belongs to the Flaviviridae family, includes ssRNA+ viruses responsible for infectious diseases in pigs, cattle, sheep, goats and other domestic and wild ruminants. Like most of the RNA viruses, pestivirus has high genome variability with practical consequences on disease epidemiology, diagnosis and control. In addition to the officially recognized species in the genus Pestivirus, such as BVDV‐1, BVDV‐2, BDV and CSFV, other pestiviruses have been detected. Furthermore, most of the ruminant pestiviruses show low or absent species specificity observed in serological tests and are able to infect multiple species. Particularly, small ruminants are receptive hosts of the most heterogeneous group of pestiviruses. The aim of this study was to carry out the molecular characterization of pestiviruses isolated from sheep and goats in Sicily, Italy. Phylogenetic analysis of two viral genomic regions (a fragment of 5′‐UTR and the whole N^pro regions) revealed the presence of different pestivirus genotypes in the analysed goat and sheep herds. Two of five viral isolates were clustered with BVDV‐1d viruses, a strain widespread in Italy, but never reported in Sicily. The other three isolates formed a distinct cluster with high similarity to Tunisian isolates, recently proposed as a new pestivirus species. This represents the first evidence for Tunisian‐like pestivirus presence in small ruminants in Italy. Furthermore, one of the isolates was collected from a goat, representing the first isolation of Tunisian‐like pestivirus from this species.     

Genetic and phylogenetic analysis of reemerged novel Seneca Valley virus strains in Guangdong province, 2017

From June to July 2017, six Seneca Valley virus (SVV ) strains were isolated from swine herds exhibiting SVV ‐associated porcine idiopathic vesicular disease (PIVD ) in Guangdong province, China. Complete genomic sequences of these six newly identified strains were genetically and phylogenetically analysed. The results revealed that these six SVV strains were genetically closely related to USA /GBI 29/2015 and notably distinct from all previous Chinese strains, indicating the reemergence of new SVV strains in Guangdong province.