Equine Influenza Virus in Asia: Phylogeographic Pattern and Molecular Features Reveal Circulation of an Autochthonous Lineage.

Equine influenza virus (EIV) causes severe acute respiratory disease in horses. Currently, the strains belonging to the H3N8 subtype are divided into two clades, Florida clade 1 (FC1) and Florida clade 2 (FC2), which emerged in 2002. Both FC1 and FC2 clades were reported in Asian and Middle East countries in the last decade. In this study, we described the evolution, epidemiology, and molecular characteristic of the EIV lineages, with focus on those detected in Asia from 2007 to 2017. The full genome phylogeny showed that FC1 and FC2 constituted separate and divergent lineages, without evidence of reassortment between the clades. While FC1 evolved as a single lineage, FC2 showed a divergent event around 2004 giving rise to two well-supported and coexisting sublineages, European and Asian. Furthermore, two different spread patterns of EIV in Asian countries were identified. The FC1 outbreaks were caused by independent introductions of EIV from the Americas, with the Asian isolates genetically similar to the contemporary American lineages. On the other hand, the FC2 strains detected in Asian mainland countries conformed to an autochthonous monophyletic group with a common ancestor dated in 2006 and showed evidence of an endemic circulation in a local host. Characteristic aminoacidic signature patterns were detected in all viral proteins in both Asian-FC1 and FC2 populations. Several changes were located at the top of the HA1 protein, inside or near antigenic sites. Further studies are needed to assess the potential impact of these antigenic changes in vaccination programs.IMPORTANCE The complex and continuous antigenic evolution of equine influenza viruses (EIVs) remains a major hurdle for vaccine development and the design of effective immunization programs. The present study provides a comprehensive analysis showing the EIV evolutionary dynamics, including the spread and circulation within the Asian continent and its relationship to global EIV populations over a 10-year period. Moreover, we provide a better understanding of EIV molecular evolution in Asian countries and its consequences on the antigenicity. The study underscores the association between the global horse movement and the circulation of EIV in this region. Understanding EIV evolution is imperative in order to mitigate the risk of outbreaks affecting the horse industry and to help with the selection of the viral strains to be included in the formulation of future vaccines.

Genetic linkage of capsid protein-encoding RNA segments in group A equine rotaviruses.

Rotavirus virions are formed by three concentric protein layers that enclose the 11 dsRNA genome segments and the viral proteins VP1 and VP3. Interactions amongst the capsid proteins (VP2, VP6, VP7 and VP4) have been described to play a major role in viral fitness, whilst restricting the reassortment of the genomic segments during co-infection with different rotavirus strains. In this work we describe and characterize the linkage between VP6 and VP7 proteins based on structural and genomic analyses of group A rotavirus strains circulating in Argentinean horses. Strains with the VP7 genotype G3 showed a strong association with the VP6 genotype I6, whilst strains with G14 were associated with the I2 genotype. Most of the differences on the VP6 and VP7 proteins were observed in interactive regions between the two proteins, suggesting that VP6 : VP7 interactions may drive the co-evolution and co-segregation of their respective gene segments.

Molecular Epidemiology of Rotavirus A in Calves: Evolutionary Analysis of a Bovine G8P[11] Strain and Spatio-Temporal Dynamics of G6 Lineages in the Americas.

Rotavirus A (RVA) causes diarrhea in calves and frequently possesses the G6 and P[5]/P[11] genotypes, whereas G8 is less common. We aimed to compare RVA infections and G/P genotypes in beef and dairy calves from major livestock regions of Argentina, elucidate the evolutionary origin of a G8 strain and analyze the G8 lineages, infer the phylogenetic relationship of RVA field strains, and investigate the evolution and spatio-temporal dynamics of the main G6 lineages in American countries. Fecal samples (n = 422) from diarrheic (beef, 104; dairy, 137) and non-diarrheic (beef, 78; dairy, 103) calves were analyzed by ELISA and semi-nested multiplex RT-PCR. Sequencing, phylogenetic, phylodynamic, and phylogeographic analyses were performed. RVA infections were more frequent in beef (22.0%) than in dairy (14.2%) calves. Prevalent genotypes and G6 lineages were G6(IV)P[5] in beef (90.9%) and G6(III)P[11] (41.2%) or mixed genotypes (23.5%) in dairy calves. The only G8 strain was phylogenetically related to bovine and artiodactyl bovine-like strains. Re-analyses inside the G8 genotype identified G8(I) to G8(VIII) lineages. Of all G6 strains characterized, the G6(IV)P[5](I) strains from "Cuenca del Salado" (Argentina) and Uruguay clustered together. According to farm location, a clustering pattern for G6(IV)P[5] strains of beef farms was observed. Both G6 lineage strains together revealed an evolutionary rate of 1.24 × 10-3 substitutions/site/year, and the time to the most recent common ancestor was dated in 1853. The most probable ancestral locations were Argentina in 1981 for G6(III) strains and the USA in 1940 for G6(IV) strains. The highest migration rates for both G6 lineages together were from Argentina to Brazil and Uruguay. Altogether, the epidemiology, genetic diversity, and phylogeny of RVA in calves can differ according to the production system and farm location. We provide novel knowledge about the evolutionary origin of a bovine G8P[11] strain. Finally, bovine G6 strains from American countries would have originated in the USA nearly a century before its first description.

Complete molecular genome analyses of equine rotavirus A strains from different continents reveal several novel genotypes and a largely conserved genotype constellation.

In this study, the complete genome sequences of seven equine group A rotavirus (RVA) strains (RVA/Horse-tc/GBR/L338/1991/G13P[18], RVA/Horse-wt/IRL/03V04954/2003/G3P[12] and RVA/Horse-wt/IRL/04V2024/2004/G14P[12] from Europe; RVA/Horse-wt/ARG/E30/1993/G3P[12], RVA/Horse-wt/ARG/E403/2006/G14P[12] and RVA/Horse-wt/ARG/E4040/2008/G14P[12] from Argentina; and RVA/Horse-wt/ZAF/EqRV-SA1/2006/G14P[12] from South Africa) were determined. Multiple novel genotypes were identified and genotype numbers were assigned by the Rotavirus Classification Working Group: R9 (VP1), C9 (VP2), N9 (NSP2), T12 (NSP3), E14 (NSP4), and H7 and H11 (NSP5). The genotype constellation of L338 was unique: G13-P[18]-I6-R9-C9-M6-A6-N9-T12-E14-H11. The six remaining equine RVA strains showed a largely conserved genotype constellation: G3/G14-P[12]-I2/I6-R2-C2-M3-A10-N2-T3-E2/E12-H7, which is highly divergent from other known non-equine RVA genotype constellations. Phylogenetic analyses revealed that the sequences of these equine RVA strains are related distantly to non-equine RVA strains, and that at least three lineages exist within equine RVA strains. A small number of reassortment events were observed. Interestingly, the three RVA strains from Argentina possessed the E12 genotype, whereas the three RVA strains from Ireland and South Africa possessed the E2 genotype. The unusual E12 genotype has until now only been described in Argentina among RVA strains collected from guanaco, cattle and horses, suggesting geographical isolation of this NSP4 genotype. This conserved genetic configuration of equine RVA strains could be useful for future vaccine development or improvement of currently used equine RVA vaccines.

Zoonotic RVA: State of the Art and Distribution in the Animal World.

Rotavirus species A (RVA) is a pathogen mainly affecting children under five years old and young animals. The infection produces acute diarrhea in its hosts and, in intensively reared livestock animals, can cause severe economic losses. In this study, we analyzed all RVA genomic constellations described in animal hosts. This review included animal RVA strains in humans. We compiled detection methods, hosts, genotypes and complete genomes. RVA was described in 86 animal species, with 52% (45/86) described by serology, microscopy or the hybridization method; however, strain sequences were not described. All of these reports were carried out between 1980 and 1990. In 48% (41/86) of them, 9251 strain sequences were reported, with 28% being porcine, 27% bovine, 12% equine and 33% from several other animal species. Genomic constellations were performed in 80% (32/40) of hosts. Typical constellation patterns were observed in groups such as birds, domestic animals and artiodactyls. The analysis of the constellations showed RVA's capacity to infect a broad range of species, because there are RVA genotypes (even entire constellations) from animal species which were described in other studies. This suggests that this virus could generate highly virulent variants through gene reassortments and that these strains could be transmitted to humans as a zoonotic disease, making future surveillance necessary for the prevention of future outbreaks.

Evolution of Animal South American RVA Told by the NSP4 Gene E12 Genotype.

Rotavirus A (RVA) possesses a genome of 11 double-stranded (ds) RNA segments, and each segment encodes one protein, with the exception of segment 11. NSP4 is a non-structural multifunctional protein encoded by segment 10 that defines the E-genotype. From the 31 E-genotypes described, genotype E12 has been described in Argentina, Uruguay, Paraguay, and Brazil in RVA strains infecting different animal species and humans. In this work, we studied the evolutionary relationships of RVA strains carrying the E12 genotype in South America using phylogenetic and phylodynamic approaches. We found that the E12 genotype has a South American origin, with a guanaco (Lama guanicoe) strain as natural host. Interestingly, all the other reported RVA strains carrying the E12 genotype in equine, bovine, caprine, and human strains are related to RVA strains of camelid origin. The evolutionary path and genetic footprint of the E12 genotype were reconstructed starting with the introduction of non-native livestock species into the American continent with the Spanish conquest in the 16th century. The imported animal species were in close contact with South American camelids, and the offspring were exposed to the native RVA strains brought from Europe and the new RVA circulating in guanacos, resulting in the emergence of new RVA strains in the current lineages' strongly species-specific adaption. In conclusion, we proposed the NSP4 E12 genotype as a genetic geographic marker in the RVA strains circulating in different animal species in South America.

A new molecular method for the rapid subtyping of bovine herpesvirus 1 field isolates.

Bovine herpesvirus 1 (BoHV-1) causes several clinical syndromes in cattle worldwide. There are 3 subtypes of BoHV-1: 1.1, 1.2a, and 1.2b. Several molecular methods are commonly used in the detection and characterization of BoHV-1. Among them, restriction endonuclease analysis (REA) and single-nucleotide polymorphism (SNP) analysis of the complete viral genome allow classification of BoHV-1 into different subtypes. However, developing countries need simpler and cheaper screening assays for routine testing. We designed a standard multiplex PCR followed by a REA assay allowing straightforward subclassification of all BoHV-1 isolates tested into 1.1, 1.2a, and 1.2b subtypes based on the analysis of fragment length polymorphism. Our standard multiplex PCR-REA was used to analyze 33 field strains of BoHV-1 isolated from various tissues. The assay confirmed the subtype identified previously by REA. In addition, non-polymorphic or undigested fragments were sequenced in order to confirm the mutation affecting the RE HindIII site. Our PCR-REA method is an affordable and rapid test that will subtype all BoHV-1 strains.

Phylogenetic Analyses of Rotavirus A from Cattle in Uruguay Reveal the Circulation of Common and Uncommon Genotypes and Suggest Interspecies Transmission.

Uruguay is one of the main exporters of beef and dairy products, and cattle production is one of the main economic sectors in this country. Rotavirus A (RVA) is the main pathogen associated with neonatal calf diarrhea (NCD), a syndrome that leads to significant economic losses to the livestock industry. The aims of this study are to determine the frequency of RVA infections, and to analyze the genetic diversity of RVA strains in calves in Uruguay. A total of 833 samples from dairy and beef calves were analyzed through RT-qPCR and sequencing. RVA was detected in 57.0% of the samples. The frequency of detection was significantly higher in dairy (59.5%) than beef (28.4%) calves (p < 0.001), while it did not differ significantly among calves born in herds that were vaccinated (64.0%) or not vaccinated (66.7%) against NCD. The frequency of RVA detection and the viral load were significantly higher in samples from diarrheic (72.1%, 7.99 log10 genome copies/mL of feces) than non-diarrheic (59.9%, 7.35 log10 genome copies/mL of feces) calves (p < 0.005 and p = 0.007, respectively). The observed G-types (VP7) were G6 (77.6%), G10 (20.7%), and G24 (1.7%), while the P-types were P[5] (28.4%), P[11] (70.7%), and P[33] (0.9%). The G-type and P-type combinations were G6P[11] (40.4%), G6P[5] (38.6%), G10P[11] (19.3%), and the uncommon genotype G24P[33] (1.8%). VP6 and NSP1-5 genotyping were performed to better characterize some strains. The phylogenetic analyses suggested interspecies transmission, including transmission between animals and humans.

Anti-VP6 VHH: An Experimental Treatment for Rotavirus A-Associated Disease.

Species A Rotaviruses (RVA) remain a leading cause of mortality in children under 5 years of age. Current treatment options are limited. We assessed the efficacy of two VP6-specific llama-derived heavy chain antibody fragments (VHH) -2KD1 and 3B2- as an oral prophylactic and therapeutic treatment against RVA-induced diarrhea in a neonatal mouse model inoculated with virulent murine RVA (ECw, G16P[16]I7). Joint therapeutic administration of 2KD1+3B2 (200 µg/dose) successfully reduced diarrhea duration, RVA infection severity and virus shedding in feces. While the same dose of 2KD1 or 3B2 (200 µg) significantly reduced duration of RVA-induced diarrhea, 2KD1 was more effective in diminishing the severity of intestinal infection and RVA shedding in feces, perhaps because 2KD1 presented higher binding affinity for RVA particles than 3B2. Neither prophylactic nor therapeutic administration of the VHH interfered with the host's humoral immune response against RVA. When 2KD1 (200 µg) was administered after diarrhea development, it also significantly reduced RVA intestinal infection and fecal shedding. Host antibody responses against the oral VHH treatment were not detected, nor did viral escape mutants. Our findings show that oral administration of anti-VP6 VHH constitute, not only an effective prophylactic treatment against RVA-associated diarrhea, but also a safe therapeutic tool against RVA infection, even once diarrhea is present. Anti-VP6 VHH could be used complementary to ongoing vaccination, especially in populations that have shown lower immunization efficacy. These VHH could also be scaled-up to develop pediatric medication or functional food like infant milk formulas that might help treat RVA diarrhea.

Epidemiological and virological findings during multiple outbreaks of equine influenza in South America in 2012.

BACKGROUND: In 2012, equine influenza (EI) virus was confirmed as the cause of outbreaks of respiratory disease in horses throughout South America. In Uruguay and Argentina, hundreds of vaccinated thoroughbred horses in training and racing facilities were clinically affected. OBJECTIVE: To characterise the EI viruses detected during the outbreak in Uruguay and Argentina. METHODS: Virus was detected in nasopharyngeal swabs by a pan-reactive influenza type A real-time RT-PCR. The nucleotide sequence of the HA1 gene was determined and analysed phylogenetically using mega 5 software. Amino acid sequences alignments were constructed and virus was antigenically characterised with specific ferret antisera. Paired serum samples were tested by haemagglutination inhibition and single radial haemolysis. RESULTS: The diagnosis of EIV was confirmed by real-time RT-PCR, virus isolation and serological testing. The phylogenetic analysis of HA1 gene sequences of 18 EI viruses indicated that all of them belong to clade 1 of the Florida sublineage of the American lineage and are closely related to viruses isolated in the United States in 2012. The HA1 of viruses identified in horses in racing facilities in Maroñas, Uruguay, and in Palermo, Argentina, displayed 100% amino acid sequence identity and were identical to that of a virus isolated in Dubai in 2012, from vaccinated endurance horses recently imported from Uruguay. CONCLUSIONS: The surveillance data reported illustrate the international spread of EI viruses and support the recommendations of the OIE expert surveillance panel to include viruses of the Florida sublineage in vaccines.

The first caprine rotavirus detected in Argentina displays genomic features resembling virus strains infecting members of the Bovidae and Camelidae.

Rotavirus group A (RVA) is a major cause of diarrhea in humans and young animals including small ruminants. The purpose of this study was to identify RVA in dairy goat kids, and to characterize the complete genomic constellation and genetic relatedness with other RVA strains. Four out of twenty fecal samples from diarrheic and non-diarrheic goat kids were positive for RVA by ELISA. A representative sample was selected for further genome analyses. The RVA strain RVA/Goat-wt/ARG/0040/2011/G8P[1] displayed the following genomic constellation: G8-P[1]-I2-R5-C2-M2-A3-N2-T6-E12-H3, reminiscent to guanaco and other bovine-like RVA strains detected in Argentina. Phylogenetic analyses revealed that most of the genome segments had a rather close relatedness with RVA strains typically obtained from cattle, sheep, South American camelids and goats. Interestingly, strain 0040 possessed the R5 and E12 genotypes which have up to date only been found in different animal species from Argentina. Overall, these findings suggest that strain 0040 could represent a typical goat RVA genome constellation similar to those previously found in other animal species within the order Artiodactyla.

Molecular characterization of equine rotaviruses circulating in Argentinean foals during a 17-year surveillance period (1992-2008).

P[12]G3 and P[12]G14 equine rotaviruses (ERVs) are epidemiologically important in horses. In Argentina, the prevalent ERV strains have been historically P[12]G3. The aim of this study was the detection and characterization of ERV strains circulating in foals in Argentina during a 17-year study (1992-2008). Additionally, the gene sequences of VP7, VP4 and NSP4 encoding genes of representative Argentinean ERV strains were determined and phylogenetic analyses were performed to elucidate the evolutionary relationships of the ERV strains in Argentina. ERVs were detected in 165 (21%) out of 771 diarrheic stool samples, which corresponded to 45 (39%) of 116 outbreaks from the surveyed thoroughbred horse farms. From the positive cases, 51% (n=23) were G3, 33% (n=15) were G14, 4% (n=2) represented a G3+G14 mixed infection and 11% (n=5) of the cases could not be characterized. G3 ERV was detected during the entire period, while G14 ERV was first detected in 2000 and increased its incidence specially in 2006 and 2007. All the analyzed strains belonged to the VP4 P[12] genotype, except for one G3 case which belonged to the P[3] genotype, constituting the first report of a P[3]G3 ERV strain. Phylogenetic analysis of VP7 protein revealed that the G3 Argentinean ERV strains clustered with ERVs from Ireland, while the G14 Argentinean ERV strains formed a distinct cluster within the G14 genotype. The VP4 of the P[12] ERV strains clustered with P[12] strains from Ireland and France. The NSP4 of the Argentinean ERV strains clustered with the NSP4 genotype E12, along with those of guanaco and bovine strains from Argentina, suggesting the a close evolutionary relationship among these Argentinean strains. The results of this study showed changes in the incidence of G3 and G14 during the studied period. The increase in the frequency of G14 ERV, not included in the vaccine, in the second half of the period, may have implications for vaccine design.