Genomic analysis of an acute gastroenteritis outbreak caused by rotavirus C in Hebei, China.

Rotavirus group C is an important cause of sporadic cases and outbreaks of gastroenteritis worldwide. Whole-Genome sequences of human rotavirus C (RVC) in public databases are limited. We performed genome sequencing to analyze a RVC outbreak of acute gastroenteritis in China. Samples from 22 patients were screened for pathogens using RT-PCR, and six samples were positive for rotavirus. Whole-Genome sequencing analysis showed that the outbreak strain SJZ217 belongs to the G4-P[2]-I2-R2-C2-M3-A2-N2-T2-E2-H2 genotype and shares almost identical genomic sequences with Chungnam isolated in Korea. Phylogenetic analysis revealed strain SJZ217 also fell into a cluster with rotavirus C strains from Japan and Europe. Reassortment in the VP4 fragment was observed. These results helped to understand the genetic diversity and possible spread of RVC strains.

Complex Evolutionary Dynamics of H5N8 Influenza A Viruses Revealed by Comprehensive Reassortment Analysis.

Influenza A viruses (IAVs) circulate among different species and have the potential to cause significant pandemics in humans. This study focuses on reassortment events in the H5N8 subtype of IAV, which poses a serious threat to public health due to its high pathogenicity in birds and potential for cross-species transmission. We retrieved 2359 H5N8 IAV sequences from GISAID, and filtered and analyzed 442 complete genomic sequences for reassortment events using pairwise distance deviation matrices (PDDMs) and pairwise distance correspondence plots (PDCPs). This detailed case study of specific H5N8 viruses revealed previously undescribed reassortment events, highlighting the complex evolutionary history and potential pandemic threat of H5N8 IAVs.

The resurgence of influenza A/H3N2 virus in Australia after the relaxation of COVID-19 restrictions during the 2022 season.

This study retrospectively analyzed the genetic characteristics of influenza A H3N2 (A/H3N2) viruses circulating in New South Wales (NSW), the Australian state with the highest number of influenza cases in 2022, and explored the phylodynamics of A/H3N2 transmission within Australia during this period. Sequencing was performed on 217 archived specimens, and A/H3N2 evolution and spread within Australia were analyzed using phylogenetic and phylodynamic methods. Hemagglutinin genes of all analyzed NSW viruses belonged to subclade 3C.2a1b.2a.2 and clustered together with the 2022 vaccine strain. Complete genome analysis of NSW viruses revealed highly frequent interclade reassortments between subclades 3C.2a1b.2a.2 and 3C.2a1b.1a. The estimated earliest introduction time of the dominant subgroup 3C.2a1b.2a.2a.1 in Australia was February 22, 2022 (95% highest posterior density: December 19, 2021-March 13, 2022), following the easing of Australian travel restrictions, suggesting a possible international source. Phylogeographic analysis revealed that Victoria drove the transmission of A/H3N2 viruses across the country during this season, while NSW did not have a dominant role in viral dissemination to other regions. This study highlights the importance of continuous surveillance and genomic characterization of influenza viruses in the postpandemic era, which can inform public health decision-making and enable early detection of novel strains with pandemic potential.

Identification of a cooperative effect between amino acids 169 and 174 in the rotavirus NSP4 double-layered particle-binding domain.

Segmented RNA viruses are capable of exchanging genome segments via reassortment as a means of immune evasion and to maintain viral fitness. Reassortments of single-genome segments are common among group A rotaviruses. Multiple instances of co-reassortment of two genome segments, GS6(VP6) and GS10(NSP4), have been documented in surveillance. Specifically, a division between NSP4 genotypes has been observed in the NSP4 double-layered particle (DLP)-binding domain. A previously hypothesized mechanism for this co-reassortment has been suggested to be the interaction between VP6 and NSP4 during DLP transport from viroplasms for particle maturation. In this study, we used sequence analysis, RNA secondary structure prediction, molecular dynamics and reverse genetics to form a hypothesis regarding the role of the NSP4 DLP-binding domain. Sequence analysis showed that the polarity of NSP4 DLP-binding domain amino acids 169 and 174 is clearly divided between E1 and E2 NSP4 genotypes. Viruses with E1 NSP4s had 169A/I or 169S/T with 174S. E2 NSP4s had 169R/K and 174A. RNA secondary structure prediction showed that mutation in both 545 (aa169) and 561 (aa174) causes global structure remodelling. Molecular dynamics showed that the NSP4/VP6 interaction stability is increased by mutating both aa positions 169 and 174. Using reverse genetics, we showed that an R169I mutation alone does not prevent rescue. Conversely, 174A to 174S prevented rescue, and rescue could be returned by combining 174S with 169I. When compared to rSA11 NSP4-wt, both rSA11 NSP4-R169I and rSA11 NSP4-R169I/A174S had a negligible but significant reduction in titre at specific time points. This study suggests that amino acid 174 of NSP4 may be essential in maintaining the VP6/NSP4 interaction required for DLP transport. Our results suggest that maintenance of specific polarities of amino acids at positions 169 and 174 may be required for the fitness of rotavirus field strains.

Evolution and biological characteristics of H11 avian influenza viruses isolated from migratory birds and pigeons.

The emergence of novel avian influenza reassortants in wild birds in recent years is a public health concern. However, the viruses that circulate in migratory birds are not fully understood. In this study, we summarized and categorized global H11 avian influenza viruses and reported that waterfowl and shorebirds are the major reservoirs of the identified H11 viruses. The surveillance data of the 35,749 faecal samples collected from wild bird habitats in eastern China over the past seven years revealed a low prevalence of H11 viruses in birds, with a positive rate of 0.067% (24 isolates). The phylogenetic analysis of the twenty viruses indicated that H11 viruses have undergone complex reassortment with viruses circulating in waterfowl and shorebirds. These tested viruses do not acquire mammalian adaptive mutations in their genomes and preferentially bind to avian-type receptors. Experimental infection studies demonstrated that the two tested H11N9 viruses of wild bird origin replicated and transmitted more efficiently in ducks than in chickens, whereas the pigeon H11N2 virus isolated from a live poultry market was more adapted to replicate in chickens than in ducks. In addition, some H11 isolates replicated efficiently in mice and caused body weight loss but were not lethal. Our study revealed the role of waterfowl and shorebirds in the ecology and evolution of H11 viruses and the potential risk of introducing circulating H11 viruses into ducks or chickens, further emphasizing the importance of avian influenza surveillance at the interface of migratory birds and poultry.

The PB2 I714S mutation influenced mammalian adaptation of the H3N2 canine influenza virus by interfering with nuclear import efficiency and RNP complex assembly.

Avian influenza viruses (AIVs) are the origin of multiple mammal influenza viruses. The genetic determinants of AIVs adapted to humans have been widely elucidated, however, the molecular mechanism of cross-species transmission and adaptation of AIVs to canines are still poorly understood. In this study, two H3N2 influenza viruses isolated from a live poultry market (A/environment/Guangxi/13431/2018, GX13431) and a swab sample from a canine (A/canine/Guangdong/0601/2019, GD0601) were used to investigate the possible molecular basis that determined H3N2 AIV adapting to canine. We found that GD0601 exhibited more robust polymerase activity in cells and higher pathogenicity in mice compared with its evolution ancestor H3N2 AIV GX13431. A series of reassortments of the ribonucleoprotein (RNP) complex showed that the PB2 subunit was the crucial factor that conferred high polymerase activity of GD0601, and the substitution of I714S in the PB2 subunit of GD0601 attenuated the replication and pathogenicity in mammal cells and the mouse model. Mechanistically, the reverse mutation of I714S in the PB2 polymerase subunit which was identified in AIV GX13431 reduced the nuclear import efficiency of PB2 protein and interfered with the interactions of PB2-PA/NP that affected the assembly of the viral RNP complex. Our study reveals amino acid mutation at the position of 714 in the nuclear localization signal (NLS) area in PB2 plays an important role in overcoming the barrier from poultry to mammals of the H3N2 canine influenza virus and provides clues for further study of mammalian adaptation mechanism of AIVs.

Identification of multiple inter- and intra-genotype reassortment mammalian orthoreoviruses from Japanese black cattle in a beef cattle farm.

Mammalian orthoreoviruses (MRVs), belonging to the genus Orthoreovirus in the family Spinareoviridae, possess a double-stranded RNA segmented genome. Due to the segmented nature of their genome, MRVs are prone to gene reassortment, which allows for evolutionary diversification. Recently, a genotyping system for each MRV gene segment was proposed based on nucleotide differences. In the present study, MRVs were isolated from the fecal samples of Japanese Black cattle kept on a farm in Japan. Complete genome sequencing and analysis of 41 MRV isolates revealed that these MRVs shared almost identical sequences in the L1, L2, L3, S3, and S4 gene segments, while two different sequences were found in the S1, M1, M2, M3, and S2 gene segments. By plaque cloning, at least six genetic constellation patterns were identified, indicating the occurrence of multiple inter- (S1 and M2) and intra- (M1, M3, and S2) reassortment events. This paper represents the first report describing multiple reassortant MRVs on a single cattle farm. These MRV gene segments exhibited sequence similarity to those of MRVs isolated from cattle in the U.S. and China, rather than to MRVs previously isolated in Japan. Genotypes consisting solely of bovine MRVs were observed in the L1, M1, and M2 segments, suggesting that they might have evolved within the cattle population.

Emergence of a novel pathogenic porcine G1P[7] rotavirus in China.

Among group A rotaviruses (RVAs), the G1 genotype is the main genotype causing diarrhea in children, but it has rarely been reported in pigs. During our epidemiological investigation, we detected G1P[7] rotavirus infection in piglets across several provinces in China and then isolated a porcine G1P[7] rotavirus strain (CN1P7). Sequencing revealed that the virus constellation was G1-P[7]-I5-R1-C1-M1-A8-N1-T1-E1-H1. Phylogenetic analyses revealed that CN1P7 most likely emerged due to genetic reassortment among porcine, human, giant panda and dog rotavirus strains. In vivo experiments were conducted on two-day-old piglets, which revealed that the CN1P7 strain was pathogenic to piglets. The virus was shed through the digestive tract and respiratory tract. In addition to the intestine, the CN1P7 strain displayed extraintestinal tropisms in piglets. Histopathological analysis revealed that the lung and small intestine were the targets of CN1P7. This study is the first to explore the molecular and pathogenic characterization of a pig-origin G1P[7] rotavirus.

Phylogeography and reassortment patterns of human influenza A viruses in sub-Saharan Africa.

The role of sub-Saharan Africa in the global spread of influenza viruses remains unclear due to insufficient spatiotemporal sequence data. Here, we analyzed 222 codon-complete sequences of influenza A viruses (IAVs) sampled between 2011 and 2013 from five countries across sub-Saharan Africa (Kenya, Zambia, Mali, Gambia, and South Africa); these genomes were compared with 1209 contemporaneous global genomes using phylogeographical approaches. The spread of influenza in sub-Saharan Africa was characterized by (i) multiple introductions of IAVs into the region over consecutive influenza seasons, with viral importations originating from multiple global geographical regions, some of which persisted in circulation as intra-subtype reassortants for multiple seasons, (ii) virus transfer between sub-Saharan African countries, and (iii) virus export from sub-Saharan Africa to other geographical regions. Despite sparse data from influenza surveillance in sub-Saharan Africa, our findings support the notion that influenza viruses persist as temporally structured migrating metapopulations in which new virus strains can emerge in any geographical region, including in sub-Saharan Africa; these lineages may have been capable of dissemination to other continents through a globally migrating virus population. Further knowledge of the viral lineages that circulate within understudied sub-Saharan Africa regions is required to inform vaccination strategies in those regions.

Incompatible packaging signals and impaired protein functions hinder reassortment of bat H17N10 or H18N11 segment 7 with human H1N1 influenza A viruses.

Novel bat H17N10 and H18N11 influenza A viruses (IAVs) are incapable of reassortment with conventional IAVs during co-infection. To date, the underlying mechanisms that inhibit bat and conventional IAV reassortment remain poorly understood. Herein, we used the bat influenza M gene in the PR8 H1N1 virus genetic background to determine the molecular basis that restricts reassortment of segment 7. Our results showed that NEP and M1 from bat H17N10 and H18N11 can interact with PR8 M1 and NEP, resulting in mediating PR8 viral ribonucleoprotein (vRNP) nuclear export and formation of virus-like particles with single vRNP. Further studies demonstrated that the incompatible packaging signals (PSs) of H17N10 or H18N11 M segment led to the failure to rescue recombinant viruses in the PR8 genetic background. Recombinant PR8 viruses (rPR8psH18M and rPR8psH17M) containing bat influenza M coding region flanked with the PR8 M PSs were rescued but displayed lower replication in contrast to the parental PR8 virus, which is due to a low efficiency of recombinant virus uncoating correlating with the functions of the bat M2. Our studies reveal molecular mechanisms of the M gene that hinder reassortment between bat and conventional IAVs, which will help to understand the biology of novel bat IAVs. IMPORTANCE: Reassortment is one of the mechanisms in fast evolution of influenza A viruses (IAVs) and responsible for generating pandemic strains. To date, why novel bat IAVs are incapable of reassorting with conventional IAVs remains completely understood. Here, we attempted to rescue recombinant PR8 viruses with M segment from bat IAVs to understand the molecular mechanisms in hindering their reassortment. Results showed that bat influenza NEP and M1 have similar functions as respective counterparts of PR8 to medicating viral ribonucleoprotein nuclear export. Moreover, the incompatible packaging signals of M genes from bat and conventional IAVs and impaired bat M2 functions are the major reasons to hinder their reassortment. Recombinant PR8 viruses with bat influenza M open reading frames were generated but showed attenuation, which correlated with the functions of the bat M2 protein. Our studies provide novel insights into the molecular mechanisms that restrict reassortment between bat and conventional IAVs.

Surveillance of avian influenza viruses in Hebei Province of China from 2021 to 2023: Identification of a novel reassortant H3N3.

Avian influenza remains a global public health concern for its well-known point mutation and genomic segment reassortment, through which plenty of serum serotypes are generated to escape existing immune protection in animal and human populations. Some occasional cases of human infection of avian influenza viruses (AIVs) since 2020 posed a potential pandemic risk through human-to-human transmission. Both east-west and north-south migratory birds fly through and linger in the Hebei Province of China as a stopover habitat, providing an opportunity for imported AIVs to infect the local poultry and for viral gene reassortment to generate novel stains. In this study, we collected more than 6000 environmental samples (mostly feces) in Hebei Province from 2021 to 2023. Samples were screened using real-time RT-PCR, and virus isolation was performed using the chick embryo culture method. We identified 10 AIV isolates, including a novel reassortant H3N3 isolate. Sequencing analysis revealed these AIVs are highly homologous to those isolated in the Yellow River Basin. Our findings supported that AIVs keep evolving to generate new isolates, necessitating a continuous risk assessment of local avian influenza in wild waterfowl in Hebei, China.

Detection of Dengue Virus 1 and Mammalian Orthoreovirus 3, with Novel Reassortments, in a South African Family Returning from Thailand, 2017.

In July 2017, a family of three members, a 46-year-old male, a 45-year-old female and their 8-year-old daughter, returned to South Africa from Thailand. They presented symptoms consistent with mosquito-borne diseases, including fever, headache, severe body aches and nausea. Mosquito bites in all family members suggested recent exposure to arthropod-borne viruses. Dengue virus 1 (Genus Orthoflavivirus) was isolated (isolate no. SA397) from the serum of the 45-year-old female via intracerebral injection in neonatal mice and subsequent passage in VeroE6 cells. Phylogenetic analysis of this strain indicated close genetic identity with cosmopolitan genotype 1 DENV1 strains from Southeast Asia, assigned to major lineage K, minor lineage 1 (DENV1I_K.1), such as GZ8H (99.92%) collected in November 2018 from China, and DV1I-TM19-74 isolate (99.72%) identified in Bangkok, Thailand, in 2019. Serum samples from the 46-year-old male yielded a virus isolate that could not be confirmed as DENV1, prompting unbiased metagenomic sequencing for virus identification and characterization. Illumina sequencing identified multiple segments of a mammalian orthoreovirus (MRV), designated as Human/SA395/SA/2017. Genomic and phylogenetic analyses classified Human/SA395/SA/2017 as MRV-3 and assigned a tentative genotype, MRV-3d, based on the S1 segment. Genomic analyses suggested that Human/SA395/SA/2017 may have originated from reassortments of segments among swine, bat, and human MRVs. The closest identity of the viral attachment protein σ1 (S1) was related to a human isolate identified from Tahiti, French Polynesia, in 1960. This indicates ongoing circulation and co-circulation of Southeast Asian and Polynesian strains, but detailed knowledge is hampered by the limited availability of genomic surveillance. This case represents the rare concurrent detection of two distinct viruses with different transmission routes in the same family with similar clinical presentations. It highlights the complexity of diagnosing diseases with similar sequelae in travelers returning from tropical areas.

The evolution, complexity, and diversity of swine influenza viruses in China: A hidden public health threat.

Swine influenza viruses (SIVs), including H1N1, H1N2, and H3N2, have spread throughout the global pig population. Potential pandemics are a concern with the recent sporadic cross-species transmission of SIVs to humans. We collected 1421 samples from Guangdong, Fujian, Henan, Yunnan and Jiangxi provinces during 2017-2018 and isolated 29 viruses. These included 21H1N1, 5H1N2, and 3H3N2 strains. Genome analysis showed that the domestic epidemic genotypes of H1N1 were mainly G4 and G5 reassortant EA swine H1N1. These genotypes have a clear epidemic advantage. Two strains were Clade 6B.1 pdm/09H1N1, suggesting a possible pig-to-human transmission route. Notably, three new H1N2 genotypes were identified using the genomic backbones of G4 or G5 viruses for recombination. The identification of various subtypes and genotypes highlight the complexity and diversity of SIVs in China and need for continuous monitoring of SIV evolution to assess the risks and prepare for potential influenza pandemics.

Red knots in Europe: a dead end host species or a new niche for highly pathogenic avian influenza?

The 2020/2021 epidemic in Europe of highly pathogenic avian influenza virus (HPAIV) of subtype H5 surpassed all previously recorded European outbreaks in size, genotype constellations and reassortment frequency and continued into 2022 and 2023. The causative 2.3.4.4b viral lineage proved to be highly proficient with respect to reassortment with cocirculating low pathogenic avian influenza viruses and seems to establish an endemic status in northern Europe. A specific HPAIV reassortant of the subtype H5N3 was detected almost exclusively in red knots (Calidris canutus islandica) in December 2020. It caused systemic and rapidly fatal disease leading to a singular and self-limiting mass mortality affecting about 3500 birds in the German Wadden Sea, roughly 1 % of the entire flyway population of islandica red knots. Phylogenetic analyses revealed that the H5N3 reassortant very likely had formed in red knots and remained confined to this species. While mechanisms of virus circulation in potential reservoir species, dynamics of spill-over and reassortment events and the roles of environmental virus sources remain to be identified, the year-round infection pressure poses severe threats to endangered avian species and prompts adaptation of habitat and species conservation practices.

Swine Influenza Viruses Isolated from 2019 to 2022 in Shandong Province, China, Exemplify the Dominant Genotype.

Swine influenza viruses (SIVs) have been circulating in swine globally and are potential threats to human health. During the surveillance of SIVs in Shandong Province, China, from 2019 to 2022, 21 reassortant G4 genotype Eurasian avian-like (EA) H1N1 subtypes containing genes from the EA H1N1 (HA and NA), 2009 pandemic (pdm/09) H1N1 virus (PB2, PB1, PA, NP, and M), and classical swine (CS) H1N1 (NS) lineages were isolated. The analysis of the key functional amino acid sites in the isolated viruses showed that two mutation sites (190D and 225E) that preferentially bind to the human α2-6 sialic acid receptor were found in HA. In PB2, three mutation sites (271A, 590S, and 591R) that may increase mammalian fitness and a mutation site (431M) that increases pathogenicity in mice were found. A typical human signature marker that may promote infection in humans, 357K, was found in NP. The viruses could replicate efficiently in mouse lungs and turbinates, and one of the H1N1 isolates could replicate in mouse kidneys and brains without prior adaption, which indicates that the viruses potentially pose a threat to human health. Histopathological results showed that the isolated viruses caused typical bronchopneumonia and encephalitis in mice. The results indicate that G4 genotype H1N1 has potential transmissibility to humans, and surveillance should be enhanced, which could provide important information for assessing the pandemic potential of the viruses.

Novel Genotypes of Highly Pathogenic Avian Influenza H5N1 Clade 2.3.4.4b Viruses, Germany, November 2023.

Several subtypes and many different genotypes of highly pathogenic avian influenza viruses of subtype H5 clade 2.3.4.4b have repeatedly caused outbreaks in Germany. Four new highly pathogenic avian influenza genotypes emerged in November 2023 after reassortment with low pathogenicity precursors, replacing genotype BB, which had dominated in Europe since 2022.

Genomic diversity and evolutionary dynamics of Influenza A viruses in Colombian swine: implications for one health surveillance and control.

Influenza A viruses (IAV) impose significant respiratory disease burdens in both swine and humans worldwide, with frequent human-to-swine transmission driving viral evolution in pigs and highlighting the risk at the animal-human interface. Therefore, a comprehensive One Health approach (interconnection among human, animal, and environmental health) is needed for IAV prevention, control, and response. Animal influenza genomic surveillance remains limited in many Latin American countries, including Colombia. To address this gap, we genetically characterized 170 swine specimens from Colombia (2011-2017). Whole genome sequencing revealed a predominance of pandemic-like H1N1 lineage, with a minority belonging to H3N2 and H1N2 human seasonal-like lineage and H1N1 early classical swine lineages. Significantly, we have identified reassortant and recombinant viruses (H3N2, H1N1) not previously reported in Colombia. This suggests a broad genotypic viral diversity, likely resulting from reassortment between classical endemic viruses and new introductions established in Colombia's swine population (e.g. the 2009 H1N1 pandemic). Our study highlights the importance of a One Health approach in disease control, particularly in an ecosystem where humans are a main source of IAV to swine populations, and emphasizes the need for continued surveillance and enhanced biosecurity measures. The co-circulation of multiple subtypes in regions with high swine density facilitates viral exchange, underscoring the importance of monitoring viral evolution to inform vaccine selection and public health policies locally and globally.

Characterization of Influenza D Virus Reassortant Strain in Swine from Mixed Pig and Beef Farm, France.

Influenza D virus was isolated from pigs on a mixed pig and beef farm in France. Investigation suggested bull-to-pig transmission and spread among pigs. The swine influenza D virus recovered was a reassortant of D/660 and D/OK lineages. Reported mutations in the receptor binding site might be related to swine host adaptation.

Genomic characterization of highly pathogenic avian influenza A H5N1 virus newly emerged in dairy cattle.

In March 2024, the emergence of highly pathogenic avian influenza (HPAI) A (H5N1) infections in dairy cattle was detected in the United Sates for the first time. We genetically characterize HPAI viruses from dairy cattle showing an abrupt drop in milk production, as well as from two cats, six wild birds, and one skunk. They share nearly identical genome sequences, forming a new genotype B3.13 within the 2.3.4.4b clade. B3.13 viruses underwent two reassortment events since 2023 and exhibit critical mutations in HA, M1, and NS genes but lack critical mutations in PB2 and PB1 genes, which enhance virulence or adaptation to mammals. The PB2 E627 K mutation in a human case associated with cattle underscores the potential for rapid evolution post infection, highlighting the need for continued surveillance to monitor public health threats.

Rift Valley Fever Phlebovirus Reassortment Study in Sheep.

Rift Valley fever (RVF) in ungulates and humans is caused by a mosquito-borne RVF phlebovirus (RVFV). Live attenuated vaccines are used in livestock (sheep and cattle) to control RVF in endemic regions during outbreaks. The ability of two or more different RVFV strains to reassort when co-infecting a host cell is a significant veterinary and public health concern due to the potential emergence of newly reassorted viruses, since reassortment of RVFVs has been documented in nature and in experimental infection studies. Due to the very limited information regarding the frequency and dynamics of RVFV reassortment, we evaluated the efficiency of RVFV reassortment in sheep, a natural host for this zoonotic pathogen. Co-infection experiments were performed, first in vitro in sheep-derived cells, and subsequently in vivo in sheep. Two RVFV co-infection groups were evaluated: group I consisted of co-infection with two wild-type (WT) RVFV strains, Kenya 128B-15 (Ken06) and Saudi Arabia SA01-1322 (SA01), while group II consisted of co-infection with the live attenuated virus (LAV) vaccine strain MP-12 and a WT strain, Ken06. In the in vitro experiments, the virus supernatants were collected 24 h post-infection. In the in vivo experiments, clinical signs were monitored, and blood and tissues were collected at various time points up to nine days post-challenge for analyses. Cell culture supernatants and samples from sheep were processed, and plaque-isolated viruses were genotyped to determine reassortment frequency. Our results show that RVFV reassortment is more efficient in co-infected sheep-derived cells compared to co-infected sheep. In vitro, the reassortment frequencies reached 37.9% for the group I co-infected cells and 25.4% for the group II co-infected cells. In contrast, we detected just 1.7% reassortant viruses from group I sheep co-infected with the two WT strains, while no reassortants were detected from group II sheep co-infected with the WT and LAV strains. The results indicate that RVFV reassortment occurs at a lower frequency in vivo in sheep when compared to in vitro conditions in sheep-derived cells. Further studies are needed to better understand the implications of RVFV reassortment in relation to virulence and transmission dynamics in the host and the vector. The knowledge learned from these studies on reassortment is important for understanding the dynamics of RVFV evolution.