Reassortant Strains Research Articles

Surveillance of human Group A rotavirus in Ningxia, China (2015-2021): Emergence and prevalence of G9P[8]-E2 and G3P[8]-E2 genotypes.

Group A rotaviruses (RVA) are the primary pathogens of acute gastroenteritis. Currently, two live attenuated RVA vaccines, LLR and RotaTeq, have been introduced into mainland China but are not included in the national immunization program. Because of the unknown genetic evolution of group A rotavirus in an all-age population in Ningxia, China, we monitored the epidemiological characteristics and circulating genotypes of RVA as a reference for developing vaccine strategies. We conducted seven years of consecutive surveillance of RVA based on stool samples from patients with acute gastroenteritis in sentinel hospitals in Ningxia, China, from 2015 to 2021. Reverse transcription quantitative polymerase chain reaction(RT-qPCR) was used to detect RVA in stool samples. Genotyping and phylogenetic analysis of VP7, VP4 and NSP4 genes were performed by reverse transcription-polymerase chain reaction(RT-PCR) and nucleotide sequence determination. RVA was detected in 16.58% (1436/8662) of 8662 stool samples. The positive rates were 7.17% (201/2805) and 21.09% (1235/5857) in adults and children, respectively. The most affected age group was infants and children aged 12-23months, with a positive rate of 29.53% (p<0.05). A significant winter/spring seasonality was observed. 23.29% positive rate in 2020 was the highest in 7years (p<0.05). The region with the highest positive rate in the adult group was Yinchuan, and the children's group was Guyuan. A total of 9 genotype combinations were found to be distributed in Ningxia. The dominant genotype combinations in this region gradually changed from G9P[8]-E1, G3P[8]-E1, G1P[8]-E1 to G9P[8]-E1, G9P[8]-E2, and G3P[8]-E2 during these seven years. Rare strains (e.g., G9P[4]-E1, G3P[9]-E3 and G1P[8]-E2) were occasionally detected during the study. During the study period, changes in the significant RVA circulating genotype combinations and the emergence of reassortment strains were observed, particularly the emergence and prevalence of G9P[8]-E2, G3P[8]-E2 reassortants in the region. These results indicate the importance of continuous monitoring of the molecular evolution and recombination characteristics of RVA, and should not be limited to G/P genotyping but should consider multi-gene fragment co-analysis and whole genome sequencing.

Genomic diversity and evolution analysis of severe fever with thrombocytopenia syndrome in East Asia from 2010 to 2022.

Conducting an up-to-date analysis on the genomic diversity and evolution patterns of severe fever with thrombocytopenia syndrome virus (SFTSV) is crucial for elucidating the underlying mechanisms of its emergency and pathogenicity, as well as assessing the extent of its threat to public health. Complete genome sequences of SFTSV were obtained from GenBank until December 19, 2022. A thorough phylogenetic analysis was conducted using comprehensive bioinformatics methods to estimate the genomic diversity and evolution. The phylogenetic classification of SFTSV strains yielded seven lineages (A-G) for each genome segment. SFTSV displayed notable variations in evolutionary patterns among different regions and segments, without a linear accumulation of nucleotide substitutions within segments and regions. The comprehensive analysis revealed 54 recombination events and 17 reassortment strains, including the first discovery of recombination events involving sea-crossing and species-crossing. Selection analysis identified three positive sites (2, 671, 1353) in RNA-dependent RNA polymerase, three positive sites (22, 298, 404) in glycoprotein, and two positive sites (9, 289) in nonstructural protein. No positive selection sites were found in nucleoprotein. Our study unveiled the existence of multiple evolutionary forces influencing SFTSV, contributing to its increasing genetic diversity, which had the potential to modify its antigenicity and pathogenicity. Furthermore, our study highlights the importance of tracking the spread of SFTSV across regions and species.

Current Knowledge on Epizootic Haemorrhagic Disease in China.

Epizootic haemorrhagic disease (EHD) is an infectious, non-contagious viral disease of ruminants caused by epizootic haemorrhagic disease virus (EHDV) and is transmitted by insects of the genus Culicoides. In 2008, EHD was listed on the World Organization for Animal Health (WOAH) list of notifiable terrestrial and aquatic animal diseases. This article reviews the distribution of EHD in China and relevant studies and proposes several suggestions for the prevention and control of EHD. There have been reports of positivity for serum antibodies against EHDV-1, EHDV-2, EHDV-5, EHDV-6, EHDV-7, EHDV-8 and EHDV-10 in China. Strains of EHDV-1, -5, -6, -7, -8 and -10 have been isolated, among which the Seg-2, Seg-3 and Seg-6 sequences of serotypes -5, -6, -7 and -10 belong to the eastern topotype. The emergence of western topotype Seg-2 in EHDV-1 strains indicates that EHDV-1 strains in China are reassortant strains of the western and eastern topotypes. A novel serotype strain of EHDV named YNDH/V079/2018 was isolated in 2018. Chinese scholars have successfully expressed the EHDV VP7 protein and developed a variety of ELISA detection methods, including antigen capture ELISA and competitive ELISA. A variety of EHDV nucleic acid detection methods, including RT-PCR and qRT-PCR, have also been developed. LAMP and the liquid chip detection technique are also available. To prevent and control EHD, several suggestions for controlling EHD transmission have been proposed based on the actual situation in China, including controlling the number of Culicoides, reducing contact between Culicoides and hosts, continued monitoring of EHDV and Culicoides in different areas of China and further development and application of basic and pioneering research related to EHD prevention and control.

On the nature of picobirnaviruses.

The picobirnaviruses (Picobirnaviridae, Picobirnavirus, PBVs) are currently thought to be animal viruses, as they are usually found in animal stool samples. However, no animal model or cell culture for their propagation has yet been found. In 2018, a hypothetical assumption about PBVs belonging to prokaryotic viruses was put forward and experimentally substantiated. This hypothesis is based on the presence of Shine-Dalgarno sequences in the genome of all PBVs before three reading frames (ORF) at the ribosomal binding site, with which the prokaryotic genome is saturated, while in the eukaryotic genome such regions occur with low frequency. The genome saturation with the Shine-Dalgarno sequences, as well as the preservation of this saturation in the progeny, according to scientists, allows us to attribute PBVs to prokaryotic viruses. On the other hand, there is a possibility that PBVs belong to viruses of eukaryotic hosts - fungi or invertebrates, since PBV-like sequences similar to the genome of fungal viruses from the families of mitoviruses and partitiviruses have been identified. In this regard, the idea arose that, in terms of reproduction mode, PBVs resemble fungal viruses. The divergence of views on the true PBV host(s) has sparked discussions among scientists and required further research to elucidate their nature. The review highlights the results of the search for a PBV host. The reasons for the occurrence of atypical sequences among the PBV genome sequences that use an alternative mitochondrial code of lower eukaryotes (fungi and invertebrates) for the translation of viral RNA-dependent RNA polymerase (RdRp) instead of the standard genetic code are analyzed. The purpose of the review was to collect arguments in support of the hypothesis about the phage nature of PBVs and to find the most realistic explanation of the reasons for identifying non-standard genomic sequences for PBVs. Based on the hypothesis about the genealogical relationship of PBVs with RNA viruses from other families with similar segmented genomes, such as Reoviridae, Cystoviridae, Totiviridae and Partitiviridae, virologists support the assumption of a decisive role in the origin of atypical PBV-like reassortment strains between PBVs and viruses of the listed families. The collected arguments given in this review indicate a high probability of a phage nature of PBVs. The data presented in the review show that the belonging of PBV-like progeny to prokaryotic or eukaryotic viruses is determined not only by its genome saturation level with a prokaryotic motif, standard or mitochondrial genetic code. The primary structure of the gene encoding the viral capsid protein responsible for the presence or absence of specific proteolytic properties of the virus that determine its ability for independent horizontal transmission into new cells may also be a decisive factor.

Predominance of DS‐1‐like G8P[8] rotavirus reassortant strains in children hospitalized with acute gastroenteritis in Thailand, 2018–2020

AbstractRotavirus A (RVA) is an important cause of acute gastroenteritis (AGE) in children. This study aims to investigate the molecular epidemiology of RVA in children hospitalized with AGE in Chiang Rai, Thailand in 2018–2020 by reverse transcription polymerase chain reaction. Of 302 samples, RVA was detected in 11.6% (35 samples): 11.3% (19/168) in 2018–2019 and 11.9% (16/134) in 2019–2020. G8P[8] was the predominant genotype at 68.4% in 2018–2019 and 81.2% in 2019–2020. G1P[8] (15.8%), G2P[4] (5.3%), G3P[8] (10.5%) in 2018–2019, and G9P[8] (18.8%) in 2019–2020 were also detected. Whole‐genome analysis of G8P[8] revealed a DS‐1‐like genetic backbone: G8‐P[8]‐I2‐R2‐C2‐M2‐A2‐N2‐T2‐E2‐H2. Phylogenetically, the VP7 genes of G8P[8] clustered in a major lineage with previously published 51 DS‐1‐like G8P[8] reference strains, closely related to 13 G8P[8] strains from Thailand and China. These G8P[8] strains contained two unique amino acid substitutions (A125S and N147D) in the VP7 antigenic epitopes. In addition, the VP1 and NSP2 genes of G8P[8] clustered in lineages separated from the DS‐1‐like G8P[8] reference strains with a high genetic divergence but were closely related to G1P[8], G2P[4], G3P[8], or G9P[8]. Several amino acid differences were observed in the VP7 and VP8* antigenic epitopes of G8P[8] compared with RVA vaccine strains. Homology modeling confirmed that these different amino acid residues were located on the surface‐exposed area of the structure. Taken together, the genetic analysis clearly defines the Chiang Rai DS‐1‐like G8P[8] strains as a novel reassortant strain that might have evolved genetically through reassortment events and consequently received their VP1 and NSP2 genes from locally cocirculating‐RVA genotypes.

Development of reassortant influenza vaccines: classical reassortment or reverse genetics?

An important feature of influenza vaccines, which distinguishes them from other immunobiological preparations, is that they have no fixed composition. Due to the constant influenza virus antigenic variability, production facilities require timely supply with relevant vaccine strains undoable due to the lack of proper method for the convenient, rapid and uninterrupted development of vaccine strains. Among the licensed influenza vaccines, classical inactivated and live influenza vaccines hold a special place. They are based on reassortant vaccine strains obtained by crossing currently circulating influenza virus with the so-called donor strain (cold-adapted attenuation donor for live influenza vaccines or high yield donor for inactivated vaccines). Vaccine strains for licensed live attenuated influenza vaccines are reassortants with the so-called 6:2 genome formula two genes encoding hemagglutinin and neuraminidase (HA and NA) belong to the current epidemic virus, and six genes encoding internal proteins (PB2, PB1, PA, NP, M and NS) to cold-adapted master donor virus. There is a very limited number of donors of attenuation. In Russia, there are cold-adapted viruses A/Leningrad/134/17/57 (H2N2) and B/USSR/60/69; in the USA (MedImmune) there are viruses A/Ann Arbor/6/60ca (H2N2) and B/Ann Arbor/1/66ca. MedImmune produces vaccine strains using reverse genetics technique. For other countries, this approach for obtaining vaccines is limited due to the need to purchase a license from the patent holders. In Russia, genetic manipulations with strains for the seasonal live influenza vaccine are not yet allowed; reassortants for the Russian live influenza vaccine are created only by classical reassortment in embryonated chicken eggs. Vaccine candidates for the inactivated influenza vaccine are prepared by the classical reassortment method, the requirements for them are more flexible and allow to use diverse genes combinations from wild type virus and master donor virus. High-yielding viruses such as A/PR/8/34 (H1N1), A/Texas/1/77 (H3N2), B/Lee/40 and some others are used as donors of internal genes. Unfortunately, the classical reassortment method does not always allow to promptly obtain a reassortant virus with a 6:2 genome formula. This is hindered by a number of reasons, ranging from the unique properties of a certain epidemic virus ending up with the constellation of genes. The reverse genetics method based on plasmids is an alternative approach to create reassortant vaccine strains allowing to reliably and quickly obtain reassortant viruses of a set 6:2 genome formula. However, this method also has certain weaknesses. This review discusses the advantages and disadvantages of development of conventional influenza vaccine candidates by reverse genetics and classical reassortment in developing chick embryos.

OPTIMIZATION OF CULTIVATION CONDITIONS FOR RECOMBINANT INFLUENZA VIRUSES EXPRESSING IMMUNODOMINANT M. BOVIS PROTEINS

The use of recombinant viral vectors is one of the promising approaches to the creation of a new generation of vaccines against bovine tuberculosis. For the prevention of tuberculosis in cattle, two reassortant recombinant strains of influenza A virus expressing mycobacterial proteins ESAT-6 and TB-10.4 were constructed based on the method of reverse genetics. As a result of optimization of virus cultivation conditions, the cytopathic effect of the virus was recorded on the Vero cell culture. It has been established that the maximum accumulation of viruses is noted when cultivating in a Vero cell culture using a serum-free DMEM medium at a temperature of (37 ± 0.5) ºС, with access to 5% carbon dioxide. The peak of the biological activity of the recombinant strains for both samples reached at 72 hours of the study and lasted for 96 hours. The biological activity of FLU NS_ESAT 6 was 7.50 ± 0.08 lg TCD50/cm3 , and for FLU NS_TB10.4 was 7,75 ± 0,08 lg TCD50/cm3. The results obtained are the basis for the development of viral biomass for work on the creation of an experimental series of vaccines against bovine tuberculosis.

Reassortant viruses threatening fish aquaculture

AbstractAquaculture provides more than half of fish destined for human consumption worldwide. In aquaculture, infectious diseases triggered by viruses are amongst the major cause of mortality of farmed fish. The cohabitation of different virus strains in the same geographical area opens the possibility to natural reassortment. Virus reassortment is a characteristic recombination process of segmented viruses in which different viruses merge their genome by the shuffling of complete segments. This is possible when two or more viral strains are cohabitating into a single host, boosting their probability to concatenate their viral cycles, getting to pack segments from distinct parental strains. Cohabitation of different viruses in a geographical area, and therefore the possibility of natural reassortment, has been aided by the increment in the transport of eggs and larvae between facilities around the world. Reassortant viruses have been described to show special tropisms for fish species opposed from those affected by parental virus strains, entailing an emerging threat for aquaculture but also for wild populations. To date, outbreaks of reassortant strains of infectious pancreatic necrosis virus, nodavirus (NNV), infectious salmon anaemia virus and tilapia lake virus have been described. Unfortunately, only the characterization of reassortant NNV has been explored. The aim of this review is compiling the information available concerning reassortant viruses affecting fish species of relevance for aquaculture highlighting their potential impact for the sector. Additionally, recent research advances in host‐NNV interaction are also included.

Surveillance, isolation and genomic characterization of Pteropine orthoreovirus of probable bat origin among patients with acute respiratory infection in Malaysia.

Pteropine orthoreovirus (PRV), an emerging bat-borne virus, has been linked to cases of acute respiratory infections (ARI) in humans. The prevalence, epidemiology and genomic diversity of PRV among ARI of unknown origin were studied. Among 632 urban outpatients tested negative for all known respiratory viruses, 2.2% were PRV-positive. Patients mainly presented with moderate to severe forms of cough, sore throat and muscle ache, but rarely with fever. Phylogenetic analysis revealed that over 90% of patients infected with the Melaka virus (MelV)-like PRV, while one patient infected with the Pulau virus previously found only in fruit bats. Human oral keratinocytes and nasopharyngeal epithelial cells were susceptible to clinical isolates of PRV, including the newly isolated MelV-like 12MYKLU1034. Whole genome sequence of 12MYKLU1034 using Nanopore technique revealed a novel reassortant strain. Evolutionary analysis of the global PRV strains suggests the continuous evolution of PRV through genetic reassortment among PRV strains circulating in human, bats and non-human primate hosts, creating a spectrum of reassortant lineages with complex evolutionary characteristics. In summary, the role of PRV as a common etiologic agent of ARI is evident. Continuous monitoring of PRV prevalence, pathogenicity and diversity among human and animal hosts is important to trace the emergence of novel reassortants.

A Characterization and an Evolutionary and a Pathogenicity Analysis of Reassortment H3N2 Avian Influenza Virus in South China in 2019-2020.

Seasonal H3N2 influenza virus has always been a potential threat to public health. The reassortment of the human and avian H3N2 influenza viruses has resulted in major influenza outbreaks, which have seriously damaged human life and health. To assess the possible threat of the H3N2 avian influenza virus to human health, we performed whole-genome sequencing and genetic evolution analyses on 10 H3N2 field strains isolated from different hosts and regions in 2019-2020 and selected representative strains for pathogenicity tests on mice. According to the results, the internal gene cassettes of nine strains had not only undergone reassortment with the H1, H2, H4, H6, and H7 subtypes, which circulate in poultry and mammals, but also with H10N8, which circulates in wild birds in the natural environment. Three reassorted strains were found to be pathogenic to mice, of these one strain harboring MP from H10N8 showed a stronger virulence in mice. This study indicates that reassorted H3N2 AIVs may cross the host barrier to infect mammals and humans, thereby, necessitating persistent surveillance of H3N2 AIVs.

Prevalence and genomic characterization of rotavirus group A genotypes in piglets from southern highlands and eastern Tanzania

Animals have been identified as the potential reservoirs of rotavirus group A (RVA) for human infection. However, very little is known regarding the genotype and genomic profiles of circulating RVA in Tanzanian piglets. The rotavirus genetic diversity and genome analysis was assessed among piglets from Southern highlands and Eastern Tanzania. A total of 241 faecal samples were collected from piglets in the regions of Mbeya, Iringa, and Morogoro. RVA was detected and genotyped using reverse transcription polymerase chain reaction (RT-PCR). Sanger dideoxynucleotide cycle sequencing of the viral protein (VP) 4 and VP7 genes was afterwards performed to confirm the RT-PCR results. Selected genotypes were subjected to whole genome sequencing. The overall prevalence of RVA was 35.26% (85/241) in piglets (30.58% in Mbeya, 43.75% in Iringa and 31.16% in Morogoro). Upon genotyping, the G genotypes were G4 (26), G9 (10), G3 (6), G5 (3) and the remaining 40 were untypeable, while the P genotype, were P[6] (35), P[13] (3) and the remaining 47 were untypeable. The G4P[6] were the predominant genotype followed by G3P[6], G3P[13], G4P[13] and G5P[13] were most common genotypes combinations. On phylogenetic analysis, G4 was grouped to lineage V, sublineages VIIa and VIIc, G9 to lineage I, G5 to lineage II, G3 to lineage IV, P[6] to lineage V and sublineage Ic and the P[13] to lineage IV. We revealed amino acid differences between the circulating G4 and the G4 in the ProSystems RCE vaccine used in pigs. The whole genome reveals genomic constellation of G4-P[6]-I1-R1-C1-M1-A8-N1-T1-E1-H1, G5-P[x]-I5-R1-C1-M1-A8-N1-Tx-E1-H1, G3/G4-P[13]/P[6]-Ix-R1-C1-M1-A8-N1-T1-E1-H1, G3-P[6]-Ix-R1-C1-M1-A8-N1-Tx-E1-H1 and G9-P[x]-Ix-R1-C1-M1-Ax-N1-Tx-E1-H1. The VP7 gene of G9, the VP4 gene of P[6] and NSP4 (E1) gene of some genotypes clustered together and closely related to humans origin or porcine-human reassortant strains with nucleotide similarities ranging from 97.90% to 99.74% from neighboring countries, implying possibility intragenogroup reassortment and interspecies transmission. The higher strain diversity observed within the gene segments highlight the importance of genomic analysis and continuous monitoring of RVA genotypes. Further research is needed to determine the risk factors associated with RVA infection in Tanzanian pigs in order to properly design a control program.

Infectious bursal disease virus in Western Europe: the rise of reassortant strains as the dominant field threat

ABSTRACT Infectious bursal disease virus (IBDV) is a highly contagious birnavirus causing a burdensome immunosuppressive disease in chickens. IBDV features a remarkable antigenic, pathogenic and genetic heterogeneity, with significant implications on disease manifestation, control measures and diagnostic approaches. The recent proposals of comprehensive phylogenetic classification systems offered the ideal platform for large-scale molecular surveys, which are crucial to gather epidemiological data and inform control efforts. In this study, the IBDV scenario was investigated in most of Western Europe by considering the results of diagnostic activities performed internationally throughout 2021. In total, 470 bursal samples from nine different countries were analysed by RT–PCR targeting the VP2. When a field virus was identified, the VP1 was also characterized. Most of the 132 detected field viruses were highly homologous reassortants featuring a very virulent-like VP2 and a classical-like VP1 (genotype A3B1). Despite emerging recently, these reassortants were already signalled in several countries in North-Western Europe and associated with subclinical infections. Here, we report their further spread in the region, where they currently represent the dominant field threat. Two other IBDV types were found, one in Italy, where all the identified viruses clustered in a clade of the A3B1 genotype previously reported only in Russia and the Middle East, and the other in Portugal, where the recently characterized A9B1 genotype was confirmed to circulate. The obtained data suggest the recent occurrence of a major shift in the Western European epidemiological landscape of IBDV, stressing the importance of steady monitoring and sharing of information among different countries and laboratories. RESEARCH HIGHLIGHTS The IBDV scenario in Western Europe seems to have radically changed in recent years. IBDV reassortants were found to be the dominant field type in the region. Local circulation of two other IBDV types was detected in Italy and Portugal.

Identification of Novel Reassortant Shuni Virus Strain in Clinical Cases of Israeli Ruminants, 2020-2021.

The Shuni virus (SHUV) causes an endemic viral infection in Israel and South Africa. It belongs to the Simbu serogroup within the order Bunyavirales, family Peribunyaviridae, genus Orthobunyavirus. Recently, it has been identified in aborted cases of domestic ruminants, young cattle and horses manifesting neural signs and acute death, symptomatic cows, and in carcasses of wild animals. Moreover, SHUV was isolated and identified in humans. In this study, we describe clinical cases of SHUV infection in Israeli domestic ruminants in 2020–2021, which represented clinical manifestations of simbuviral infection including abortions, a neural lethal case in a fattening calf, and an acute symptomatic case in a beef cow. In all cases, SHUV was confirmed by complete or partial viral genome sequencing. There is a significant difference of M and L segments of the novel strains compared with those of all known SHUV strains, while the S segments have more than 99% nucleotide (nt) identity with Israeli and African “Israeli-like” strains previously circulated in 2014–2019. This indicates a reassortment origin of the strain. At the same time, M and S segment nt sequences showed about 98–99% nt identity with some South African strains collected in 2016–2018. Nevertheless, the viral origin and the geographical place of the reassortment stayed unknown.

Hemorrhagic fever with renal syndrome caused by destruction of residential area of rodent in a construction site: epidemiological investigation

BackgroundAn outbreak of hemorrhagic fever with renal syndrome (HFRS), caused by a Hantavirus, affected nine adult males in the southwest area of Xi’an in November 2020 was analyzed in this study.MethodsClinical and epidemiological data of HFRS patients in this outbreak were retrospectively analyzed. The whole genome of a hantavirus named 201120HV03xa (hv03xa for short) isolated from Apodemus agrarius captured in the construction site was sequenced and analyzed. In addition, nine HFRS patients were monitored for the IgG antibody against the HV N protein at 6 and 12 months, respectively.ResultsIn this study, inhalation of aerosolized excreta and contaminated food may be the main source of infection. Genome analysis and phylogenetic analysis showed that hv03xa is a reassortment strain of HTNV, having an S segment related to A16 of HTN 4, an M segment related to Q37 and Q10 of HTN 4, and an L segment related to prototype strain 76–118 of HTN 7. Potential recombination was detected in the S segment of hv03xa strain. The anti-HV-IgG level of all the patients persist for at least one year after infection.ConclusionsThis report documented an HFRS outbreak in Xi’an, China, which provided the basic data for epidemiological surveillance of endemic HTNV infection and facilitated to predict disease risk and implement prevention measures.

Genetics and Pathogenicity of Natural Reassortant of Infectious Bursal Disease Virus Emerging in Latvia

Infectious bursal disease virus is an immunosuppressive pathogen that, despite applied vaccination, is affecting the poultry industry worldwide. This report presents the genetic and pathotypic characterization of a natural reassortant emerging in Europe (Latvia). Genetic characterization showed that strain 25/11/Latvia/2011 represents genotype A3B1, whose segment A is derived from very virulent strains, while segment B is from the classic-like genogroup. Phylogenetic maximum likelihood inference of the B-segment sequence clustered the reassortant strain together with the US antigenic variant E strain. However, the obtained full-length sequence of 25/11/Latvia/2011 revealed that not only reassortment but also dozens of mutations shaped the unique genetic makeup. Phenotypic characterization showed no mortality and no clinical signs of disease but a severe bursa of Fabricius atrophy and splenomegaly in the convalescent birds at 10 days post infection. The results obtained indicate that the acquired genetic constellation contributed to a decrease in virulence; nevertheless, the infection causes severe damage to lymphoid organs, which can lead to impaired immune responses.

Evaluations of rationally designed rift valley fever vaccine candidate RVax-1 in mosquito and rodent models

Rift Valley fever (RVF) is a mosquito-borne zoonosis endemic to Africa and the Arabian Peninsula, which causes large outbreaks among humans and ruminants. Single dose vaccinations using live-attenuated RVF virus (RVFV) support effective prevention of viral spread in endemic countries. Due to the segmented nature of RVFV genomic RNA, segments of vaccine strain-derived genomic RNA could be incorporated into wild-type RVFV within co-infected mosquitoes or animals. Rationally designed vaccine candidate RVax-1 displays protective epitopes fully identical to the previously characterized MP-12 vaccine. Additionally, all genome segments of RVax-1 contribute to the attenuation phenotype, which prevents the formation of pathogenic reassortant strains. This study demonstrated that RVax-1 cannot replicate efficiently in orally fed Aedes aegypti mosquitoes, while retaining strong immunogenicity and protective efficacy in an inbred mouse model, which were indistinguishable from the MP-12 vaccine. These findings support further development of RVax-1 as the next generation MP-12-based vaccine for prevention of Rift Valley fever in humans and animals.

Molecular evolution and genetic characteristics of G3P[3] group A canine rotavirus isolated in Wuhan, China.

Rotaviruses can infect multiple animal species and have the potential for cross-recombination based on the segmented genome characteristics. To study the intra-host recombination and zoonotic potential of group A canine rotavirus (CRV), 438 samples were collected from domestic dogs in six animal hospitals and from stray dogs from October 2019 to May 2021 in Wuhan, China. Seven of the samples were positive (7/438) for group A CRV from which a CRV strain was successfully isolated in MA-104 cells. The genotype of the isolated strain was characterized by whole-genome sequencing showing that the genotype was group A CRV G3P[3]. According to the Rotavirus Classification Working Group (RCWG), the genomic constellation of the isolated CRV was G3-P[3]-I3-R3-C3-M3-A9-N2-T3-E3-H6, which belongs to the AU-1-like group with gene segments of AU-1-like and Cat 97-like strains. Based on the phylogenetic analysis of the 11 gene segments, we found that the different segments of the isolated group A CRV were closely related to several reassortment rotaviruses from different animal sources and bat strains. Based on the analysis of the molecular evolution and genetic characteristics, we concluded that the isolated strain might be a reassortment strain. These data further enrich our understanding of rotavirus molecular evolution and genetic characteristics in China.

Human infection of avian influenza A H3N8 virus and the viral origins: a descriptive study

The H3N8 avian influenza virus (AIV) has been circulating in wild birds, with occasional interspecies transmission to mammals. The first human infection of H3N8 subtype occurred in Henan Province, China, in April, 2022. We aimed to investigate clinical, epidemiological, and virological data related to a second case identified soon afterwards in Hunan Province, China. We analysed clinical, epidemiological, and virological data for a 5-year-old boy diagnosed with H3N8 AIV infection in May, 2022, during influenza-like illness surveillance in Changsha City, Hunan Province, China. H3N8 virus strains from chicken flocks from January, 2021, to April, 2022, were retrospectively investigated in China. The genomes of the viruses were sequenced for phylogenetic analysis of all the eight gene segments. We evaluated the receptor-binding properties of the H3N8 viruses by using a solid-phase binding assay. We used sequence alignment and homology-modelling methods to study the effect of specific mutations on the human receptor-binding properties. We also conducted serological surveillance to detect the H3N8 infections among poultry workers in the two provinces with H3N8 cases. The clinical symptoms of the patient were mild, including fever, sore throat, chills, and a runny nose. The patient's fever subsided on the same day of hospitalisation, and these symptoms disappeared 7 days later, presenting mild influenza symptoms, with no pneumonia. An H3N8 virus was isolated from the patient's throat swab specimen. The novel H3N8 virus causing human infection was first detected in a chicken farm in Guangdong Province in December, 2021, and subsequently emerged in several provinces. Sequence analyses revealed the novel H3N8 AIVs originated from multiple reassortment events. The haemagglutinin gene could have originated from H3Ny AIVs of duck origin. The neuraminidase gene belongs to North American lineage, and might have originated in Alaska (USA) and been transferred by migratory birds along the east Asian flyway. The six internal genes had originated from G57 genotype H9N2 AIVs that were endemic in chicken flocks. Reassortment events might have occurred in domestic ducks or chickens in the Pearl River Delta area in southern China. The novel H3N8 viruses possess the ability to bind to both avian-type and human-type sialic acid receptors, which pose a threat to human health. No poultry worker in our study was positive for antibodies against the H3N8 virus. The novel H3N8 virus that caused human infection had originated from chickens, a typical spillover. The virus is a triple reassortment strain with the Eurasian avian H3 gene, North American avian N8 gene, and dynamic internal genes of the H9N2 viruses. The virus already possesses binding ability to human-type receptors, though the risk of the H3N8 virus infection in humans was low, and the cases are rare and sporadic at present. Considering the pandemic potential, comprehensive surveillance of the H3N8 virus in poultry flocks and the environment is imperative, and poultry-to-human transmission should be closely monitored. National Natural Science Foundation of China, National Key Research and Development Program of China, Strategic Priority Research Program of the Chinese Academy of Sciences, Hunan Provincial Innovative Construction Special Fund: Emergency response to COVID-19 outbreak, Scientific Research Fund of Hunan Provincial Health Department, and the Hunan Provincial Health Commission Foundation.

Development and diagnostic validation of a one-step multiplex RT-PCR assay as a rapid method to detect and identify Nervous Necrosis Virus (NNV) and its variants circulating in the Mediterranean.

Nervous Necrosis Virus (NNV) represents one of the most threatening pathogens for Mediterranean aquaculture. Several NNV strains are currently co-circulating in the Mediterranean Basin with a high prevalence of the RGNNV genotype and the RGNNV/SJNNV reassortant strain and a more limited diffusion of the SJNNV genotype and the SJNNV/RGNNV reassortant. In the present study, a one-step multiplex RT-PCR (mRT-PCR) assay was developed as an easy, cost-effective and rapid diagnostic technique to detect RGNNV and the reassortant RGNNV/SJNNV strain and to distinguish them from SJNNV and the reassortant SJNNV/RGNNV strain in a single RT-PCR reaction. A unique amplification profile was obtained for each genotype/reassortant enabling their rapid identification from cell culture lysates or directly from brain tissues of suspected fish. The method’s detection limit varied between 102.3 and 103.4 TCID50 ml-1 depending on viral strains. No cross-reacitivty with viruses and bacteria frequently associated with gilthead seabream, European seabass and marine environment was observed. The mRT-PCR was shown to be an accurate, rapid and affordable method to support traditional diagnostic techniques in the diagnosis of VNN, being able to reduce considerably the time to identify the viral genotype or the involvement of reassortant strains.

Molecular surveillance of rotavirus A associated with diarrheic calves from the Republic of Korea and full genomic characterization of bovine-porcine reassortant G5P[7] strain.

Group A rotavirus (RVA) is the most common diarrhea-causing pathogen among humans and animals worldwide. Rotavirus infection in neonatal calves causes major problems in the livestock industry. This study aimed to determine the prevalence and genetic diversity of bovine rotavirus (BoRVA) infections in calves with diarrhea and to perform whole genome analysis of an unusual strain, designated as RVA/Calf-wt/KOR/KNU-GJ2/2020/G5P[7], that was detected in a 2-day-old diarrheic calf. From 459 diarrheic calves aged 1-40days, fecal samples were collected and BoRVA infections were screened using real-time RT-PCR targeting VP6 gene. BoRVA was detected in 195 (42.4%) samples and was most prevalent in calves aged 1-10days (47.2%). No significant difference in the BoRVA infection rate was observed between calves born in herds that were (42.1%) and were not (42.6%) vaccinated against BoRVA. A binomial regression analysis revealed that calves aged 1-10days (95% confidence intervals [CI]:1.18-24.34; P=0.000) and 11-20days (95% CI: 0.76-16.83, P=0.000) had a 5.37- and 3.58-fold higher BoRVA prevalence in comparison to those aged 31-40days, respectively. The RVA-positive samples were subsequently subjected to amplification of the VP7 and VP4 genes for determining G and P genotypes. Overall, 45 (23.1%, 45/195) and 63 (32.3, 63/195) sequences for VP7 and VP4 were obtained. In this study, four G and three P genotypes were identified. G6 (86.7%) was the most prevalent genotype, followed by G8 (8.9%), G10 (2.2%), and G5 (2.2%). P[5] (92.1%) was the most frequently detected, followed by P[11] (6.3%), and P[7] (1.6%). The G6P[5] (82.2%) is the most common combination found in Korean native calves with diarrhea, whereas G6P[11] (4.4%) and G10P[11] (2.2%) had relatively low prevalence. G8P[5] (8.9%) was identified for the first time in diarrheic calves in the KOR. The uncommon strain KNU-GJ2 exhibited a G5-P[7]-I5-R1-C1-M2-A1-N1-T1-E1-H1 genotype constellation possessing a typical porcine RVA backbone, with the exception of the VP3 gene, which is derived from bovine. Phylogenetically, except for VP3, ten gene segments of KNU-GJ2 were closely related to porcine, porcine-like, and reassortant bovine strains. Interestingly, the VP3-M2 gene of KNU-GJ2 clustered with bovine-like strains as well as reassortant porcine and bovine strains. Comparison of the NSP4s within a species-specific region of amino acids 131-141 demonstrated that KNU-GJ2 belonged to genotype B with porcine RVAs; however, it differed from porcine RVAs by one to three amino acids. The present study is fundamental to understanding the epidemiology and genotypes of circulating RVAs throughout the KOR and underscoring the importance of continuous monitoring and molecular characterization of RVAs circulating within animal populations for future vaccine development.