An experimental study of Bungowannah virus infection in weaner aged pigs

Animal-to-human interspecies transmission is one of the evolutionary mechanisms driving rotavirus strain diversity in humans. Although quite a few studies emanating from Africa revealed evidence of bovine-to-human rotavirus interspecies transmission, whole genome data of African bovine rotavirus strains are not yet available. To gain insight into the complete genome constellation of African bovine rotaviruses, the full genomes of three bovine rotavirus strains were extracted from stool samples collected from calves, amplified using a sequence-independent procedure, followed by 454(?) pyrosequencing. Strains RVA/Cow-wt/ZAF/1603/2007/G6P[5] and RVA/Cow-wt/ZAF/1605/2007/G6P[5] were both genotyped as G6-P[5]-I2-R2-C2-M2-A3-N2-T6-E2-H3 and were probably two variants of the same rotavirus due to their close nucleotide sequence similarity. The genotype constellation of strain RVA/Cow-wt/ZAF/1604/2007/G8P[1] was G8-P[1]-I2-R2-C2-M2-A3-N2-T6-E2-H3. The genetic relationships and phylogenetic analyses suggested that these three bovine rotavirus strains may have emerged through multiple reassortment events between bovine, giraffe and antelope rotaviruses. Due to the close relatedness of genome segments 1 (encoding VP1), 7 (NSP2), 9 (VP7) and 10 (NSP4) of strain RVA/Cow-wt/ZAF/1604/2007/G8P[1] to those of the corresponding segments of human rotaviruses, RVA strain 1604 may represent bovine strains that were transmitted to humans and possibly reassorted with human rotaviruses previously. The complete nucleotide sequences of the bovine rotavirus strains reported in this study represent the first whole genome data of bovine rotaviruses from Africa.     

Detection and genetic characterization of porcine group A rotaviruses in asymptomatic pigs in smallholder farms in East Africa: Predominance of P[8] genotype resembling human strains

Viral enteritis is a serious problem accounting for deaths in neonatal animals and humans worldwide. The absence of surveillance programs and diagnostic laboratory facilities have resulted in a lack of data on rotavirus associated diarrheas in pigs in East Africa. Here we describe the incidence of group A rotavirus (RVA) infections in asymptomatic young pigs in East Africa. Of the 446 samples examined, 26.2% (117/446) were positive for RVA. More nursing piglets (78.7%) shed RVA than weaned (32.9%) and grower (5.8%) pigs. RVA incidence was higher in pigs that were either housed_free-range (77.8%) or tethered_free-range (29.0%) than those that were free-range or housed or housed-tethered pigs. The farms with larger herd size (>10 pigs) had higher RVA prevalence (56.5%) than farms with smaller herd size (24.1–29.7%). This study revealed that age, management system and pig density significantly (p < 0.01) influenced the incidence of RVA infections, with housed_free-range management system and larger herd size showing higher risks for RVA infection. Partial (811–1604 nt region) sequence of the VP4 gene of selected positive samples revealed that different genotypes (P[6], P[8] and P[13]) are circulating in the study area with P[8] being predominant. The P[6] strain shared nucleotide (nt) and amino acid (aa) sequence identity of 84.4–91.3% and 95.1–96.9%, respectively, with known porcine and human P[6] strains. The P[8] strains shared high nt and aa sequence identity with known human P[8] strains ranging from 95.6–100% to 92–100%, respectively. The P[13] strains shared nt and aa sequence identity of 83.6–91.7% and 89.3–96.4%, respectively, only with known porcine P[13] strains. No P[8] strains yielded RNA of sufficient quality/quantity for full genome sequencing. However analysis of the full genome constellation of the P[6], two P[13] and one untypeable strains revealed that the P[6] strain (Ke-003-5) genome constellation was G26-P[6]-I5-R1-C1-M1-A8-N1-T1-E1-H1, P[13] strains (Ug-049 and Ug-453) had G5-P[13]-I5-R1-C1-M1-A8-N1-T7-E1-H1 while the untypeable strain (Ug-218) had G5-P[?]-I5-R1-C1-M1-A8-N1-T1-E1-H? In conclusion, P[6] and P[8] genotypes detected were genetically closely related to human strains suggesting the possibility of interspecies transmission. Further studies are required to determine the role of RVA in swine enteric disease burden and to determine the genetic/antigenic heterogeneity of the circulating strains for development of accurate diagnostic tools and to implement appropriate prophylaxis programs.     

Evidence for the porcine origin of equine rotavirus strain H-1

Equine group A rotavirus (RVA) strain H-1 (RVA/Horse-tc/GBR/H-1/1975/G5P9[7]) was found to have VP4, VP6-7, NSP1 and NSP4 genes of porcine origin. In order to obtain conclusive information on the exact origin and evolution of this unusual equine strain, the remaining six genes (VP1-3, NSP2-3 and NSP5 genes) of strain H-1 were analyzed in the present study. By whole genomic analysis, strain H-1 exhibited a porcine RVA-like genotype constellation (G5-P[7]-I5-R1-C1-M1-A8-N1-T1-E1-H1), different from those of typical equine RVA strains. The VP2-3 and NSP2-3 genes of strain H-1 were found to originate from porcine RVAs. On the other hand, it was difficult to pinpoint the exact origin of the VP1 and NSP5 genes of strain H-1, though phylogenetically, these genes appeared to be possibly derived from porcine or Wa-like human strains. Taken together, at least nine (VP2-4, VP6-7 and NSP1-4 genes) of the 11 gene segments of strain H-1 were found to be of porcine origin, revealing a porcine RVA-like genetic backbone. Therefore, strain H-1 is likely a porcine RVA strain that was transmitted to horses.     

Full-length genome analysis of G2, G9 and G11 porcine group A rotaviruses

Group A rotaviruses with G2 and G9 VP7 specificity are common in humans, while G11 strains have been detected only sporadically. G2, G9 and G11 rotaviruses also circulate in pigs and swine rotaviruses have been suspected of interspecies and zoonotic transmissions in numerous studies. However, the complete gene constellation of G2 and G9 porcine rotaviruses has not yet been determined. In order to start filling this gap, the genomic make up of two G2, one G9 and one G11 porcine rotavirus strains, detected in Canada in 2005–2007, was determined. With the exception of a G2P[34] strain, with E9 NSP4 type and mixed I5 + I14 VP6 type, the constellation of genomic segments was rather conserved and were closely related to prototype porcine strains in the four viruses characterized (I5-R1-C1-M1-A8-N1-T7-E1-H1). Most notably, all the viruses displayed a rare NSP3 genotype, T7, which has also been identified in rare human reassortant strains and in the reference strain RVA/Cow-tc/GBR/UK/1973/G6P[5]. This study provides crucial genetic data on these complex viruses and will help understand the origin and ecological niche of gene segments and the role played by pigs in their evolution.     

Intestinal and extra-intestinal pathogenicity of a bovine reassortant rotavirus in calves and piglets

Despite the impact of bovine group A rotaviruses (GARVs) as economically important and zoonotic pathogens, there is a scarcity of data on cross-species pathogenicity and extra-intestinal spread of bovine reassortant GARVs. During the course of characterizing the genotypes of all 11 genomic segments of bovine GARVs isolated from diarrheic calves in South Korea, a unique G6P[7] reassortant GARV strain (KJ9-1) was isolated. The strain harbors five bovine-like gene segments (VP7: G6; VP6: I2; VP1: R2; VP3: M2; NSP2: N2, and NSP4: E2), five porcine-like gene segments (VP4: P[7]; NSP1: A1; NSP3: T1, and NSP5: H1), and one human-like gene segment (VP2: C2). To investigate if this reassortant strain possessed cross-species pathogenicity in calves and piglets, and could induce viremia and extra-intestinal spread in calves, colostrum-deprived calves and piglets were experimentally inoculated with the KJ9-1 strain. The KJ9-1 strain caused severe diarrhea in experimentally infected calves with extensive intestinal villous atrophy, but replicated without causing clinical symptoms in experimentally infected piglets. By SYBR Green real-time RT-PCR, viral RNA was detected in sera of the calves at post-inoculation day (PID) 1, reaching a peak at PID3, and then rapidly decreasing from PID4. In addition, viral RNA was detected in the mesenteric lymph node, lungs, liver, choroid plexus, and cerebrospinal fluid. An immunofluorescence assay confirmed viral replication in the extra-intestinal organs and tissues of virus-inoculated calves. The data indicates that the homologous/heterologous origin of the NSP4 gene segment (E2 genotype), may play a key role in the ability to cause diarrhea in calves and piglets.     

Molecular characterization of unusual bovine group A rotavirus G8P[14] strains identified in western India: emergence of P[14] genotype

A total of 78 fecal specimens were collected from both apparently healthy (n=71) and diarrheic (n=7) cattle from an organized farm in Pune, western India in December 2007-January 2008. Three specimens tested positive for group A rotavirus (RV) by antigen capture ELISA were subjected to RT-PCR for amplification of entire coding regions of three structural (VP4, VP6 and VP7) and one nonstructural (NSP4) genes. All three strains were genotyped as G8P[14]. Phylogenetic analysis of the VP7 and VP4 genes showed clustering of the VP7 gene with G8 strains of bovine origin and VP4 gene with P[14] strains of human origin. The identification of VP6 and NSP4 genes to have I2 (subgroup I) and E2 (genotype A) specificity, respectively of bovine and human origin indicated independent segregation of genes in bovine RV strains. This study indicates circulation of a rare RV genotype, G8P[14] in western India. To our knowledge, this is the second report on RV G8[14] isolated from bovine species after bovine group A RV strain, SUN9 from Japan.     

Discovery and molecular characterization of a group A rotavirus strain detected in an Argentinean vicuña (Vicugna vicugna)

The wild vicuña (Vicugna vicugna) is one of the four species of native South American camelids (SACs) in addition to the wild guanaco, and their domesticated counterparts, alpaca and llama, respectively. Serological data have indicated the presence of group A rotaviruses (RVA) specific antibodies in all 4 members of the SAC, and so far, RVA has been detected from alpacas, llamas and guanacos. A total of 59 fecal samples from healthy wild newborn and juvenile vicuñas, raised in captivity in Jujuy, Argentina were collected and analyzed by ELISA to detect RVA antigen. Two samples (3%) were found to contain G8 RVA strains and one strain (RVA/Vicuña-wt/ARG/C75/2010/G8P[14]) was selected for further genome analyses, revealing the G8-P[14]-I2-R2-C2-M2-Ax-N2-T6-E3-Hx genotype constellation. Unfortunately, no sequence data could be obtained for NSP1 and NSP5. Except for the E3 NSP4 genotype, this partial genotype constellation is reminiscent to bovine RVA strains and bovine-like RVA strains isolated from sheep, guanaco, antelope and humans. This relationship was confirmed phylogenetically, providing further evidence of the widespread presence of this genotype constellation in animals belonging to the artiodactyls. In particular, a close phylogenetic relationship was found between C75 and guanaco RVA strain RVA/Guanaco-wt/ARG/Chubut/1999/G8P[14] for at least 5 gene segments, suggesting a partial conservation of the genotype constellation of RVA strains infecting different species of SACs, even though nowadays their natural habitats are not overlapping. The further monitoring of the sanitary health of wild newborn and juvenile vicuñas is essential to improve the management practices applied in their sustainable exploitation.     

Genetic diversity and novel combinations of G4P[19] and G9P[19] porcine rotavirus strains in Thailand

Several epidemiological studies reported the detection of rotavirus strains bearing unusual combinations of genetic background of human and porcine rotaviruses. This observation supports the hypothesis of interspecies transmission of rotaviruses in humans and pigs. The aims of this study were to investigate the genotypes and molecular characteristics of rotaviruses in piglets with diarrhea in several farms from two provinces in Thailand. A total of 207 fecal specimens collected from diarrheic piglets were screened for the presence of groups A, B, and C rotaviruses. Group A rotaviruses were detected in 41 out of 207 (19.8%) fecal specimens tested. A wide variety of G-P combination rotavirus strains were detected in this study. The G4P[6] was identified as the most prevalent genotype (39.0%), followed by G4P[23] (12.2%), G3P[23] (7.3%), G4P[19] (7.3%), G3P[6] (4.9%), G3P[13] (4.9%), G3P[19] (4.9%), G9P[13] (4.9%), G9P[19] (4.9%), G5P[6], and G5P[13] each of 2.4%. Furthermore, G5 and G9 in combinations with P-nontypeable strains were also found at each consisting of 2.4% (n = 1) of the collection. It was interesting to note that among diversified porcine rotavirus strains, novel combinations of G4P[19] and G9P[19] strains were detected for the first time in this study. Nucleotide sequences of VP4 and VP7 of these strains were closely related to human rotaviruses reported previously. The data implies that these porcine rotaviruses were probably generated in nature from the reassortment between the viruses of human and porcine origin. This study provides valuable epidemiological information and molecular characteristics of porcine rotaviruses circulating in piglets with diarrhea in northern Thailand.     

Genetic heterogeneity of wild-type G4P[6] porcine rotavirus strains detected in a diarrhea outbreak in a regularly vaccinated pig herd

Group A rotavirus (RV-A) with short electropherotype was identified by ss-PAGE in a neonatal diarrhea outbreak at a Brazilian pig farm where the sows were regularly vaccinated with a commercial vaccine containing OSU (G5P[7]) and Gottfried (G4P[6]) porcine RV-A (PoRV-A) strains. The ss-PAGE positive stool samples (n=20) were characterized as P[6] genotype by multiplex-nested-RT-PCR assay. The nucleotide analysis of the VP4 gene (VP8*) state that the viruses clustered in P[6] lineages that are also shared by RV-A strains identified in human hosts. Nucleotide analysis of the VP7 gene identified different lineages in G4 including a new lineage tentatively designated IX. The immunological pressure induced by commercial vaccine with a rotavirus containing a G4P[6] genotype of porcine origin (Gottfried strain) might have allowed the selection of PoRV-A strains with characteristics found in RV-A strains isolated of human hosts, such as P[6]-Ie and If, and promoted the selection or emergence of RV-A strains with a new lineage of the G4 genotype. The characterization of PoRV-A strains with unusual genotypes described in this study highlight the importance of surveys on the relationship between human and animal rotavirus strains.     

G and P genotype profiles of rotavirus A field strains circulating in beef and dairy cattle herds in Brazil, 2006–2015

The aim of this retrospective study was to use RT-PCR and nucleotide sequencing analysis to determine the G (VP7 gene) and P (VP4 gene) genotypes of 155 Brazilian bovine rotavirus A (RVA) wild-type strains detected in diarrheic calves from all Brazilian geographical regions from 2006 to 2015. The RVA strains evaluated belonged to the G6, G10, P[5], and P[11] genotypes. The G6P[5] genotype was prevalent (65.5%; P < 0.05) in beef, and the G10P[11] (38.4%) and G6P[11] (30.8%) genotypes were more prevalent in dairy cattle herds. The Midwest was the region with the highest number of genotyped RVA strains, where the genotypes G6, P[5], and P[11] were identified. Genotype combination G6-IV/P[5]-IX, prevalent in beef herds, and G6-III/P[11]-III or G10-IV/P[11]-III, prevalent in dairy herds, were detected. In addition, for the first time in Brazil, we detected the P[5] and P[11] genotype RVA strains that belong to lineage II and VII, respectively.     

Lapine rotaviruses of the genotype P[22] are widespread in Italian rabbitries

An epidemiological survey was carried out to investigate the distribution of the VP7 and VP4 specificities of lapine rotaviruses (LRVs) in rabbitries from different geographical regions of Italy. Almost all the strains were characterized as P[22],G3, confirming the presence of the newly-recognized rotavirus P[22] VP4 allele in Italian rabbits. Only one P[14],G3 LRV strain was identified and two samples contained a mixed (P[14] + [22],G3) rotavirus infection. All the LRV strains analyzed exhibited a genogroup I VP6 specificity and a long dsRNA electropherotype. However, one of the P[14],G3 strains possessed a super-short pattern. Altogether, these data highlight the epidemiological relevance of the P[22] LRVs in Italian rabbitries.     

Frequency of group A rotavirus with mixed G and P genotypes in bovines: predominance of G3 genotype and its emergence in combination with G8/G10 types

The present study describes the genotypic distribution of rotaviruses (RVs) in an Indian bovine population with unexpectedly higher proportions of G3 alone or in combination of G8/G10. PCR-genotyping confirmed that 39.4% (13/33) of the prevalent RVs were the G3 type while 60.6% (20/33) were dual G3G10 or G3G8 types. P typing revealed that 93.9% (31/33) of the samples were P[11] while 6.1% (2/33) possessed a dual P[1]P[11] type. Sequence analysis of the VP7 gene from G3 strains viz. B-46, 0970, and BR-133 showed that these strains had sequence identities of 90.5% to 100% with other bovine G3 strains. The highest identity (98.9% to 100%) was observed with RUBV3 bovine G3 strains from eastern India. The G3 strains (B-46, 0970, and BR-133) showed 97.5% to 98.8% sequence homologies with the Indian equine RV strain Erv-80. Phylogenetic analysis demonstrated that G3 strains clustered with bovine RUBV3 and J-63, and equine Erv-80 G3. Overall, these results confirmed that the incidence of infection by RVs with the G3 genotype and mixed genotypes in the bovine population was higher than previously predicted. This finding reinforces the importance of constantly monitoring circulating viral strains with the G3 genotype in future surveillance studies.     

Phylogenetic analysis of porcine rotavirus in Argentina: increasing diversity of G4 strains and evidence of interspecies transmission

Group A rotaviruses are one of the most frequently detected viral agents associated with neonatal diarrhea in piglets. In order to characterize rotavirus (RV) strains circulating in Argentinean swine, four porcine production farms located in Buenos Aires were studied. RV strains genotyped as P[6]G4, P[6]G8 and P[1]G6 were found in piglets under 30 days of age, without diarrhea. Phylogenetic and sequence analysis of the VP7 gene from G4 strains available in databases, reveals five porcine new lineages (III-VII) and three sublineages (VIIa-VIIc). The G4 porcine Argentinean strains were grouped with a porcine RV strain isolated in Brazil and another RV strain isolated from a child with diarrhea in Mexico, constituting an American lineage (VII). On the other hand, porcine G6 and G8 were closely related to RV's circulating in Argentinean cattle and South-American camelids, respectively. The fact that G4 porcine lineages were epidemiologically related to human strains, and G6 and G8 Argentinean porcine strains were found related to bovine and South-American camelids, respectively, suggests that pigs might play a crucial role as reservoir and generator of newly adapted emerging RV strains for human and other species.     

Molecular characterization of unusual bovine rotavirus A strains having high genetic relatedness with human rotavirus: evidence for zooanthroponotic transmission

We report here the genomic characterization of two rare rotavirus A (RVAs) G1P[11] and G9P[X] strains detected in cattle calves from two different geographical locations in India during routine rotavirus surveillance. These strains possessed unusual G types (VP7 gene) on a bovine/artiodactyl genotype constellation, G1‐P[11]‐I2‐Rx‐Cx‐Mx‐Ax‐N2‐T6‐E2‐H3 (HR‐B91) and G9‐P[X]‐I2‐Rx‐Cx‐Mx‐Ax‐N2‐T6‐E2‐H3 (WB‐H2). This is the first report on molecular characterization of G9 in cattle, and second report on G1 in cattle. The VP7 gene of HR‐B91 occupied lineage IIc within G1 while that of WB‐H2 occupied IIIb within G9 genotype. The latter was found to be very diverse from other RVA strains of G9 genotype, and this may emerge as a new genotype in due course. The study provides evidence of zooanthroponotic transmission of human G1 and G9 RVA genes to calves. Of note, the G9 genotype was found to serve as the ancestral genotype for G1. Phylogenetic analysis of remaining gene segments revealed close relatedness to artiodactyl or artiodactyl‐like human RVA strains. The findings of this study highlight the potential role of interspecies transmission and reassortment events in generating the rare rotavirus strains.     

Equine G3P[3] rotavirus strain E3198 related to simian RRV and feline/canine-like rotaviruses based on complete genome analyses

Equine group A rotavirus (RVA) strains are the most important cause of gastroenteritis in equine neonates and foals worldwide, and G3P[12] and G14P[12] are epidemiologically the most important genotypes. The genotype constellation of an unusual Argentinean G3P[3] RVA strain (RVA/Horse-wt/E3198/2008/G3P[3]) detected in fecal samples of a diarrheic foal in 2008 was shown to be G3–P[3]–I3–R3–C3–M3–A9–N3–T3–E3–H6. Each of these genotypes has been found typically in feline and canine RVA strains, and the genotype constellation is reminiscent to those of Cat97-like RVA strains. However, the phylogenetic analyses revealed only a distant relationship between E3198 and known feline, canine and feline/canine-like human RVA strains. Surprisingly, a rather close relationship was found between E3198 and simian RVA strains RVA/Simian-tc/USA/RRV/1975/G3P[3] for at least 5 gene segments. RRV is believed to be a reassortant between a bovine-like RVA strain and a RVA strains distantly related to feline/canine RVA strains. These analyses indicate that E3198 is unlikely to be of equine origin, and most likely represents a RVA interspecies transmitted virus, possibly in combination with one or more reassortments, from a feline, canine or related host species to a horse. Further studies are in progress to evaluate if this strain was a single interspecies transmission event, or if this strain started to circulate in the equine population.     

First description of group A rotavirus from fecal samples of ostriches (Struthio camelus)

This study investigated the occurrence of rotavirus infections in ostriches (Struthio camelus) reared in Northern Paraná, Brazil. Fecal (n=66) and serum (n=182) samples from nine farms located in four different cities were analyzed by silver stained-polyacrylamide gel electrophoresis (ss-PAGE), RT-PCR assay, virus isolation, and counterimmunoelectroosmophoresis (CIE). Rotavirus group A seropositivity occurred in 5.49% (10/182) of serum samples of ostriches originated from two farms. Only 9.09% (6/66) of fecal samples from ostriches with diarrhea maintained in one farm were positive by ss-PAGE, RT-PCR, and virus isolation. The G (VP7) and P (VP4) genotypes of rotavirus wild strains isolated in cell culture were determined by multiplex-nested PCR. The genotyping identified two rotavirus strains: G6P[1] and G10P[1]. In three rotavirus strains it was only possible to identify the P type; one strain being P[1] and two strains that presented the combination of P[1]+P[7]. These findings might represent the first characterization of rotavirus in ostriches, and the finding of porcine and bovine-like rotavirus genotypes in ostriches might suggest virus reassortment and possible interspecies transmission.     

Antibody response in vaccinated pregnant mares to recent G3BP[12] and G14P[12] equine rotaviruses

Background

Both the G3P[12] and the G14P[12] type of equine group A rotavirus (RVA) have recently become predominant in many countries, including Japan. G3 types are classified further into G3A and G3B. The G3A viruses have been circulating in Europe, Australia, and Argentina, and the G3B viruses have been circulating in Japan. However, only an inactivated vaccine containing a single G3BP[12] strain is commercially available in Japan. To assess the efficacy of the current vaccine against recently circulating equine RVA strains, we examined antibody responses in pregnant mares to recent G3BP[12] and G14P[12] strains by virus neutralization test.

Findings

After vaccination in five pregnant mares, the geometric mean serum titers of virus-neutralizing antibody to recent G3BP[12] strains increased 5.3- to 7.0-fold and were similar to that against homologous vaccine strain. Moreover, antibody titers to recent G14P[12] strains were also increased 3.0- to 3.5-fold.

Conclusions

These results suggest that inoculation of mares with the current vaccine should provide foals with virus-neutralizing antibodies against not only the G3BP[12] but also the G14P[12] RVA strain via the colostrum.     

Genetic and serological characterization of novel serotype G8 bovine group A rotavirus strains isolated in Japan

G8 bovine group A rotaviruses isolated in Japan were genetically and serologically characterized. The VP7 gene nucleotide and amino acid sequences revealed high identity with each other. All Japanese G8 strains were classified into the same lineage in the phylogenetic analysis based on VP7 gene sequences. Antisera to four Japanese G8 strains neutralized other G8 strains, but their neutralizing titers were between 8-fold lower and 2-fold higher than homologous strains. These results suggest that the VP7s of Japanese G8 strains have similar genetic and serologic characteristics. Observed differences in the neutralizing abilities of antisera for each strain appear to depend on differences in the P serotypes/genotypes.