Vaccine-derived Strains Research Articles

Wide circulation of type 1 vaccine-derived poliovirus strains in clinical specimens from suspected cases of poliomyelitis, their contacts and in wastewater in Madagascar since late 2020

Madagascar has faced three major outbreaks of vaccine-derived polioviruses (VDPVs) in recent decades, with VDPV type 1 reemerging in late 2020. Here, we report the molecular characterization of these cVDPV1 strains. WHO protocols were used for poliovirus detection in stool and wastewater samples. Molecular genotyping was based on the 5′ non-coding (5′NC), VP1, and 3Dpol regions. From 2020 to 2022, 92 of 5690 stool samples and 129 of 1046 wastewater samples tested positive for cVDPV1. Genetic analysis of the VP1 gene revealed 1.3%–6.1% variability compared to the Sabin strain. Most sequences showed mutations at neurovirulence attenuation sites. Phylogenetic analysis distributed strains into four genogroups originating from Southern Madagascar. All analyzed cVDPV1 strains were recombinant, containing mutated oral polio vaccine sequences in VP1 and type C enterovirus sequences in other regions. This study demonstrated that all strains were closely related during this epidemic.

Simultaneous detection of infectious bronchitis virus and avian metapneumovirus genotypes A, B, and C by multiplex RT-qPCR assay in chicken tracheal samples in Ecuador.

Respiratory RNA viruses such as Infectious bronchitis virus (IBV) and Avian metapneumovirus (aMPV), which are characterized by generating both respiratory damage and adverse effects on reproductive organs, affect poultry production economically due to high mortality rate and decrease in egg production and quality. Particularly, aMPV has three genotypes that have been reported with greater frequency in chickens: aMPV-A, aMPV-B, and aMPV-C. The present study proposes the design of a multiplex RT-qPCR assay for the simultaneous diagnosis of the 3 genotypes of interest of aMPV and IBV, followed by testing of 200 tracheal samples of vaccinated chickens with respiratory symptoms and finally a phylogenetic analysis of the sequences found. The assay detected up to 1 copy of each viral genome. The standard curves showed an efficiency between 90 and 100% in the multiplex assay and inter- and intra-assay coefficients of variation of 0.363 and 0.459, respectively and inter- and intra-assay coefficients of variation of 0.363 and 0.459, respectively. 69.5% of samples were found positive alone or in coinfection. 114 samples were positive for IBV, 13 for aMPV-A and 25 for aMPV-B. RNA of aMPV-C was no detected. The most commonly found combination was aMPV-B and IBV within 6 samples, and the least common was aMPV-A and aMPV-B in coinfection in 2 samples. The assay was specific for amplification of the genomes of the studied respiratory viruses (IBV, aMPV-A, aMPV-B, aMPV-C) as no amplification was shown from other viral genomes (ChPV, CAstV, ANV, and FAdV) or from the negative controls. Partial genomic Sanger sequencing enabled to identify circulating vaccine-derived and wild-type strains of IBV and vaccine and vaccine-derived strains of aMPV-B. In conclusion, this newly developed multiplex RT-qPCR was shown to be able to detect individual infections as well as co-infections among the respiratory viruses investigated. It was demonstrated to be a reliable and efficient tool for rapidly and safely diagnosing these infections. Furthermore, this study represents the first report of aMPV strains in Ecuadorian poultry and demonstrates the circulation of aMPV-A, aMPV-B, and GI-13 IBV strains in unvaccinated chicken populations in the country. Thus, it highlights the importance of simultaneously identifying these pathogens in greater detail and on a regular basis in Ecuador.

Polio outbreak response, Yemen.

A decrease in vaccine coverage in conflict-affected areas has placed Yemen at higher risk of polio outbreaks caused by vaccine-derived poliovirus strains. In response to polio outbreaks, the Yemeni health ministry and partners initiated multiple vaccination campaigns to deliver vaccines to children. We also implemented several measures to enhance communication, education, health promotion and hygiene, especially in camps for internally displaced people. In 2009, Yemen achieved polio-free status and maintained it until 2019. However, the ongoing political conflict since 2015, coupled with challenges in delivering the polio vaccine to conflict-affected areas, resulted in two polio outbreaks: 35 cases caused by vaccine-derived poliovirus strain 1 between 2019 and 2021, and 230 cases due to vaccine-derived poliovirus strain 2 between November 2021 and December 2022. In response to the first outbreak, by the end of 2020, we vaccinated 7.2 million children through nationwide vaccination campaigns, except in Sa'ada governorate due to a ban by the authorities. By the end of 2021, 3 800 313 children younger than 5years had received polio vaccines. For the second outbreak, by the end of 2022, 4 463 389 vaccines had been given to children younger than 10years, and 1 217 423 to those younger than 5years. Vaccination campaigns in conflict-affected areas with low vaccine coverage remain crucial in eradicating polio. Efforts are needed to reach vulnerable groups such as displaced populations. Advocacy, communication and social mobilization actions help ensure broader public inclusion and participation in vaccination efforts to prevent polio outbreaks.

Worst-case scenarios: Modeling uncontrolled type 2 polio transmission.

In May 2016, the Global Polio Eradication Initiative (GPEI) coordinated the cessation of all use of type 2 oral poliovirus vaccine (OPV2), except for emergency outbreak response. Since then, paralytic polio cases caused by type 2 vaccine-derived polioviruses now exceed 3,000 cases reported by 39 countries. In 2022 (as of April 25, 2023), 20 countries reported detection of cases and nine other countries reported environmental surveillance detection, but no reported cases. Recent development of a genetically modified novel type 2 OPV (nOPV2) may help curb the generation of neurovirulent vaccine-derived strains; its use since 2021 under Emergency Use Listing is limited to outbreak response activities. Prior modeling studies showed that the expected trajectory for global type 2 viruses does not appear headed toward eradication, even with the best possible properties of nOPV2 assuming current outbreak response performance. Continued persistence of type 2 poliovirus transmission exposes the world to the risks of potentially high-consequence events such as the importation of virus into high-transmission areas of India or Bangladesh. Building on prior polio endgame modeling and assuming current national and GPEI outbreak response performance, we show no probability of successfully eradicating type 2 polioviruses in the near term regardless of vaccine choice. We also demonstrate the possible worst-case scenarios could result in rapid expansion of paralytic cases and preclude the goal of permanently ending all cases of poliomyelitis in the foreseeable future. Avoiding such catastrophic scenarios will depend on the development of strategies that raise population immunity to type 2 polioviruses.

Immunogenicity of Catch-Up Immunization with Conventional Inactivated Polio Vaccine among Japanese Adults.

Most Japanese adults are vaccinated twice with the Sabin trivalent oral polio vaccine. Booster vaccination is recommended for Japanese travelers to polio-endemic/high-risk countries. We assessed the catch-up immunization of healthy Japanese adults aged ≥20 years with two doses of standalone conventional inactivated polio vaccine (cIPV). Immunogenicity was evaluated by serum neutralization titers (pre-booster vaccination, 4-6 weeks after each vaccination) against type 1, 2, and 3 poliovirus strains. The participants were 61 healthy Japanese adults (26 men/35 women; mean age ± standard deviation age 35.8 ± 8.0 years). Seropositivity rates (percentage of participants with anti-poliovirus antibody titers ≥1:8) pre-vaccination were 88.5%, 95.1%, and 52.5% for Sabin strains (type 1, 2, and 3); 72.1%, 93.4%, and 31.1% for virulent poliovirus strains (type 1: Mahoney; type 2: MEF-1; and type 3: Saukett); and 93.4%, 93.4%, 93.4%, and 88.5% for type 2 vaccine-derived poliovirus strains (SV3128, SV3130, 11,196, and 11,198). After one cIPV dose, all seropositivity rates increased to 98.4-100.0%. After two cIPV doses, the seropositivity rates reached 100% for all strains. cIPV was well tolerated, with no safety concerns. Catch-up immunization with standalone cIPV induced robust immune responses in Japanese adults, indicating that one booster dose boosted serum-neutralizing antibodies to many strains.

Genetic and phenotypic stability of poliovirus shed from infants who received novel type 2 or Sabin type 2 oral poliovirus vaccines in Panama: an analysis of two clinical trials

Sabin strains used in oral poliovirus vaccines (OPV) can revert to virulence and, in rare instances, cause disease or generate vaccine-derived strains leading to outbreaks in areas of low immunisation coverage. A novel OPV2 (nOPV2) was designed to stabilise the viral genome against reversion and reduce recombination events that might lead to virulent strains. In this study, we evaluated the genetic and phenotypic stability of shed poliovirus following administration of one dose of monovalent OPV2 (mOPV2) or nOPV2 to infants aged 18-22 weeks. In two similarly designed clinical trials (NCT02521974 and NCT03554798) conducted in Panama, infants aged 18-22-weeks, after immunisation with three doses of bivalent OPV (types 1 and 3) and one dose of inactivated poliovirus vaccine, were administered one or two doses of mOPV2 or nOPV2. In this analysis of two clinical trials, faecally shed polioviruses following one dose of mOPV2 or nOPV2 were isolated from stools meeting predetermined criteria related to sample timing and viral presence and quantity and assessed for nucleotide polymorphisms using next-generation sequencing. A transgenic mouse neurovirulence test was adapted to assess the effect of the possible phenotypic reversion of shed mOPV2 and nOPV2 with a logistic regression model. Of the 91 eligible samples, 86 were able to be sequenced, with 72 evaluated in the transgenic mouse assay. Sabin-2 poliovirus reverts rapidly at nucleotide 481, the primary attenuation site in domain V of the 5' untranslated region of the genome. There was no evidence of neurovirulence-increasing polymorphisms in domain V of shed nOPV2. Reversion of shed Sabin-2 virus corresponded with unadjusted paralysis rates of 47·6% at the 4 log10 50% cell culture infectious dose (CCID50) and 76·7% at the 5 log10 CCID50 inoculum levels, with rates of 2·8% for 4 log10 CCID50 and 11·8% for 5 log10 CCID50 observed for shed nOPV2 samples. The estimated adjusted odds ratio at 4·5 log10 of 0·007 (95% CI 0·002-0·023; p<0·0001) indicates significantly reduced odds of mouse paralysis from virus obtained from nOPV2 recipients compared with mOPV2 recipients. The data indicate increased genetic stability of domain V of nOPV2 relative to mOPV2, with significantly lower neurovirulence of shed nOPV2 virus compared with shed mOPV2. While this vaccine is currently being deployed under an emergency use listing, the data on the genetic stability of nOPV2 will support further regulatory and policy decision-making regarding use of nOPV2 in outbreak responses. Bill & Melinda Gates Foundation.

Experimentally controlled study indicates that the naturally occurring recombinant vaccine-like lumpy skin disease strain Udmurtiya/2019, detected during freezing winter in northern latitudes, is transmitted via indirect contact.

Lumpy skin disease (LSD) caused by LSD virus (LSDV), is a member of the poxvirus genus Capripoxvirus. It is classified as a notifiable disease by the World Organization for Animal Health (WOAH) based on its potential for rapid spread and global economic impact. Due to these characteristics, the mode of LSDV transmission has prompted intensive research efforts. Previous experimental studies using the virulent vaccine-derived recombinant LSDV strain Saratov/2017, demonstrated that this strain has the capacity for transmission in a vector-proof environment. This study demonstrated that a second novel recombinant vaccine-derived LSDV strain Udmurtiya/2019, can infect bulls in contact with diseased animals, in the absence of insect vectors. Bulls were housed in an insect proof animal biosafety level 3 facility, where half the animals were inoculated intravenously with the recombinant LSDV (Udmurtiya/2019), whilst the remaining five animals were mock-inoculated but kept in contact with the inoculated group. Both the infected / inoculated group (IN) and uninfected / incontact group (IC), were monitored for 41 days with continuous registration of body temperature, observations for clinical signs and collection of blood samples and nasal swabs for testing of LSDV presence using real-time PCR. Results indicated that cohabitation of animals from both groups was sufficient to transmit the virus from the IN to the IC-group, with the onset of clinical signs including pyrexia (~41°C) and classical LSD nodular skin lesions starting at 10 dpi for the IN group and 16 dpi for the IC-group. Additionally, the presence of LSDV genomes as well as anti-LSDV antibodies were detected in swabs, blood and serum samples from animals belonging to both groups. These results provides additional evidence of LSDV transmission in a controlled environment without direct contact between diseased and healthy animals, yet in the absence of vectors. Based on these observations, the question concerning a hypothetical relation between mutations in the virus genome and its mode of transmission gains more importance and requires additional investigations with direct comparisons between classical and novel recombinant LSDV strains.

Rapid Spread and Genetic Characterisation of a Recently Emerged Recombinant Lumpy Skin Disease Virus in Thailand.

Simple SummaryLumpy skin disease (LSD) is an economically important disease of cattle caused by LSD virus (LSDV), a member of poxviruses. It had never been found in Thailand before March 2021, but has since spread broadly to various provinces. Regional veterinarians have collected samples from the LSD cattle and submitted them for diagnosis as a part of disease surveillance during the outbreaks. Our study aimed to monitor the distribution of the outbreaks by recording the LSD cases based on clinical signs and laboratory tests up to June 2022, and characterise the causative agent virologically and genetically. Outbreak maps were created to illustrate the rapid temporal distribution of the LSD index cases in each province of Thailand. We detected two distant origins of the outbreaks. LSDV DNA was confirmed in blood, milk, and skin samples collected from sick animals by real-time PCR. LSDV was proven to be the causative virus based on serological, virological, and pathological diagnoses. By genetic analysis, the Thai LSDV is a recombinant virus derived from a vaccine strain previously appearing in China and Vietnam. Its genetic material is a mosaic hybrid genome containing the vaccine virus DNA as the backbone interspersed with DNA fragments of a field strain.The emergence of the lumpy skin disease virus (LSDV) was first detected in north-eastern Thailand in March 2021. Since then, the abrupt increase of LSD cases was observed throughout the country as outbreaks have spread rapidly to 64 out of a total of 77 provinces within four months. Blood, milk, and nodular skin samples collected from affected animals have been diagnosed by real-time PCR targeting the p32 gene. LSDV was isolated by primary lamb testis (PLT) cells, followed by Madin-Darby bovine kidney (MDBK) cells, and confirmed by immunoperoxidase monolayer assay (IPMA). Histopathology and immunohistochemistry (IHC) of a skin lesion showed inclusion bodies in keratinocytes and skin epithelial cells. Phylogenetic analyses of RPO30 and GPCR genes, and the whole genome revealed that Thai viruses were closely related to the vaccine-derived recombinant LSDV strains found previously in China and Vietnam. Recombination analysis confirmed that the Thai LSDV possesses a mosaic hybrid genome containing the vaccine virus DNA as the backbone and a field strain DNA as the minor donor. This is an inclusive report on the disease distributions, complete diagnoses, and genetic characterisation of LSDV during the first wave of LSD outbreaks in Thailand.

Rotavirus vaccine-derived cases in Belgium: Evidence for reversion of attenuating mutations and alternative causes of gastroenteritis

Since the introduction of live-attenuated rotavirus vaccines in Belgium in 2006, surveillance has routinely detected rotavirus vaccine-derived strains. However, their genomic landscape and potential role in gastroenteritis have not been thoroughly investigated. We compared VP7 and VP4 nucleotide sequences obtained from rotavirus surveillance with the Rotarix vaccine sequence. As a result, we identified 80 vaccine-derived strains in 5125 rotavirus-positive infants with gastroenteritis from 2007 to 2018. Using both viral metagenomics and reverse transcription qPCR, we evaluated the vaccine strains and screened for co-infecting enteropathogens. Among the 45 patients with known vaccination status, 39 were vaccinated and 87% received the vaccine less than a month before the gastroenteritis episode. Reconstruction of 30 near complete vaccine-derived genomes revealed 0–11 mutations per genome, with 88% of them being non-synonymous. This, in combination with several shared amino acid changes among strains, pointed at selection of minor variant(s) present in the vaccine. We also found that some of these substitutions were true revertants (e.g., F167L on VP4, and I45T on NSP4). Finally, co-infections with known (e.g., Clostridioides difficile and norovirus) and divergent or emerging (e.g., human parechovirus A1, salivirus A2) pathogens were detected, and we estimated that 35% of the infants likely had gastroenteritis due to a ‘non-rotavirus’ cause. Conversely, we could not rule out the vaccine-derived gastroenteritis in over half of the cases. Continued studies inspecting reversion to pathogenicity should monitor the long-time safety of live-attenuated rotavirus vaccines. All in all, the complementary approach with NGS and qPCR provided a better understanding of rotavirus vaccine strain evolution in the Belgian population and epidemiology of co-infecting enteropathogens in suspected rotavirus vaccine-derived gastroenteritis cases.

Viral subpopulation variability in different batches of Infectious bronchitis virus (IBV) vaccines based on GI-23 lineage: Implications for the field

The control of infectious bronchitis (IB) is largely based on routine vaccine administration, often using live-attenuated vaccines. However, their capability to replicate and be transmitted among animals and farms implies significant risks. The detection of strains genetically related to vaccines complicates the diagnostic process and understanding of the viral molecular epidemiology. Moreover, reversion to virulence and associated clinical outbreaks can occur although the underlying mechanism are often unknown. In the present study, three vaccine vials, based on IBV GI-23 lineage (also known as Variant2) were deep sequenced through Next Generation Sequencing (NGS) to investigate the presence and features of viral subpopulations. To elucidate the consequences in the field and identify potential markers suitable for a DIVA strategy, the S1 sequences of strains originating from farms in different countries were sequenced and classified based on the knowledge of their vaccination history and similarity with the applied vaccine. Although all considered vaccine batches shared the same consensus sequence, different subpopulations were identified suggesting independent and poorly constrained evolutionary processes. When compared with strains sampled from farms, the vaccine consensus sequences and the respective subpopulations clustered with vaccine strains and no genetic features were consistently shared with field strains. Therefore, if vaccine-induced outbreaks occur, they are more likely to originate from in vivo evolution rather than selection of already present subpopulations. Although some amino acid residues were most commonly detected in field or vaccine strains, no consistent marker could be identified.The occurrence of subpopulations within IBV GI-23-based vaccines and variability featuring different production batches was demonstrated. Being such a phenomenon apparently driven by random genetic drift rather than directional selection, the differentiation between field and vaccine-derived strains appears extremely challenging based on sequence analysis alone. The knowledge of farm management and vaccination history should thus be considered for a proper epidemiological investigation.

Intradermal fractional-dose inactivated polio vaccine (fIPV) adjuvanted with double mutant Enterotoxigenic Escherichia coli heat labile toxin (dmLT) is well-tolerated and augments a systemic immune response to all three poliovirus serotypes in a randomized placebo-controlled trial

Eradication of poliomyelitis globally is constrained by fecal shedding of live polioviruses, both wild-type and vaccine-derived strains, into the environment. Although inactivated polio vaccines (IPV) effectively protect the recipient from clinical poliomyelitis, fecal shedding of live virus still occurs following infection with either wildtype or vaccine-derived strains of poliovirus. In the drive to eliminate the last cases of polio globally, improvements in both oral polio vaccines (OPV) (to prevent reversion to virulence) and injectable polio vaccines (to improve mucosal immunity and prevent viral shedding) are underway. The E. coli labile toxin with two or “double” attenuating mutations (dmLT) may boost immunologic responses to IPV, including at mucosal sites. We performed a double-blinded phase I controlled clinical trial to evaluate safety, tolerability, as well as systemic and mucosal immunogenicity of IPV adjuvanted with dmLT, given as a fractional (1/5th) dose intradermally (fIPV-dmLT). Twenty-nine volunteers with no past exposure to OPV were randomized to a single dose of fIPV-dmLT or fIPV alone. fIPV-dmLT was well tolerated, although three subjects had mild but persistent induration and hyperpigmentation at the injection site. A ≥ 4-fold rise in serotype-specific neutralizing antibody (SNA) titers to all three serotypes was seen in 84% of subjects receiving fIPV-dmLT vs. 50% of volunteers receiving IPV alone. SNA titers were higher in the dmLT-adjuvanted group, but only differences in serotype 1 were significant. Mucosal immune responses, as measured by polio serotype specific fecal IgA were minimal in both groups and differences were not seen. fIPV-dmLT may offer a benefit over IPV alone. Beyond NAB responses protecting the individual, studies demonstrating the ability of fIPV-dmLT to prevent viral shedding are necessary. Studies employing controlled human infection models, using monovalent OPV post-vaccine are ongoing. Studies specifically in children may also be necessary and additional biomarkers of mucosal immune responses in this population are needed.Clinicaltrials.gov Identifer: NCT03922061.

WGS- versus ORF5-Based Typing of PRRSV: A Belgian Case Study.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of one of the most widespread and economically devastating diseases in the swine industry. Typing circulating PRRSV strains by means of sequencing is crucial for developing adequate control strategies. Most genetic studies only target the highly variable open reading frame (ORF) 5, for which an extensive database is available. In this study, we performed whole-genome sequencing (WGS) on a collection of 124 PRRSV-1 positive serum samples that were collected over a 5-year period (2015–2019) in Belgium. Our results show that (nearly) complete PRRSV genomes can be obtained directly from serum samples with a high success rate. Analysis of the coding regions confirmed the exceptionally high genetic diversity, even among Belgian PRRSV-1 strains. To gain more insight into the added value of WGS, we performed phylogenetic cluster analyses on separate ORF datasets as well as on a single, concatenated dataset (CDS) containing all ORFs. A comparison between the CDS and ORF clustering schemes revealed numerous discrepancies. To explain these differences, we performed a large-scale recombination analysis, which allowed us to identify a large number of potential recombination events that were scattered across the genome. As PRRSV does not contain typical recombination hot-spots, typing PRRSV strains based on a single ORF is not recommended. Although the typing accuracy can be improved by including multiple regions, our results show that the full genetic diversity among PRRSV strains can only be captured by analysing (nearly) complete genomes. Finally, we also identified several vaccine-derived recombinant strains, which once more raises the question of the safety of these vaccines.

Detection and Clinical Implications of Monovalent Rotavirus Vaccine-Derived Virus Strains in Children with Gastroenteritis in Alberta, Canada.

While rotavirus vaccine programs effectively protect against severe rotavirus gastroenteritis, rotavirus vaccine strains have been identified in the stool of vaccinated children and their close contacts suffering from acute gastroenteritis. The prevalence of vaccine strains, the emergence of vaccine-derived strains, and their role in acute gastroenteritis are not well studied. We developed a locked nucleic acid reverse transcription real-time PCR assay (LNA-RTqPCR) to detect the monovalent rotavirus vaccine (RV1) Rotarix nonstructural protein 2 (NSP2) in children with acute gastroenteritis and healthy controls, and validated it using sequence-confirmed RV1 strains. The association between RV1-derived strains and gastroenteritis was determined using logistic regression. The new assay exhibited 100% (95% CI 91.7%, 100%) diagnostic sensitivity and 99.4% (95% CI 96.2%, 100%) diagnostic specificity, with a detection limit of 9.86 copies/reaction and qPCR efficiency of 99.7%. Using this assay, we identified the presence of RV1-derived NSP2 sequences in 7.7% of rotavirus gastroenteritis cases and 98.6% of rotavirus-positive healthy children (94.4% had previously received the RV1). Among gastroenteritis cases, those whose stool contained RV1-derived strains had milder gastroenteritis symptoms compared to that of natural rotavirus infections. We observed no significant association between RV1-derived strains and gastroenteritis (odds ratio [OR] 0.98; 95% CI 0.60, 1.72). Our study demonstrated that the new assay is suitable for monitoring RV1-derived rotavirus strain circulation and that the RV1-derived strains are not associated with development of gastroenteritis symptoms.

Assessment of genetic changes and neurovirulence of shed Sabin and novel type 2 oral polio vaccine viruses

Sabin-strain oral polio vaccines (OPV) can, in rare instances, cause disease in recipients and susceptible contacts or evolve to become circulating vaccine-derived strains with the potential to cause outbreaks. Two novel type 2 OPV (nOPV2) candidates were designed to stabilize the genome against the rapid reversion that is observed following vaccination with Sabin OPV type 2 (mOPV2). Next-generation sequencing and a modified transgenic mouse neurovirulence test were applied to shed nOPV2 viruses from phase 1 and 2 studies and shed mOPV2 from a phase 4 study. The shed mOPV2 rapidly reverted in the primary attenuation site (domain V) and increased in virulence. In contrast, the shed nOPV2 viruses showed no evidence of reversion in domain V and limited or no increase in neurovirulence in mice. Based on these results and prior published data on safety, immunogenicity, and shedding, the nOPV2 viruses are promising alternatives to mOPV2 for outbreak responses.

Molecular epidemiology of mosquito-borne viruses at the China\u2013Myanmar border: discovery of a potential epidemic focus of Japanese encephalitis

BackgroundMosquito-based arbovirus surveillance can serve as an early warning in evaluating the status of mosquito-borne virus prevalence and thus prevent local outbreaks. Although Tengchong County in Yunnan Province—which borders Myanmar—is abundant and diverse in mosquitoes, very few mosquito-based arbovirus investigations have been conducted in the recent decade. Herein, this study aims to evaluate the presence and the diffusion of mosquito-borne pathogens, currently prevalent in this region.MethodsWe collected 9486 mosquitoes, representing eight species, with Culex tritaeniorhynchus and Anopheles sinensis as the dominant species, during high mosquito activity seasons (July–October) in Tengchong, in 2018. Samples collected from 342 pools were tested using reverse-transcription PCR to determine the species, distribution, and infection rates of virus and parasite, and further analyze their genotypes, phylogenetic relationships, infection rate, and potential pathogenicity.ResultsFifteen Japanese encephalitis virus (JEV) strains from Cx. tritaeniorhynchus pools were detected. Seven strains of insect-specific flaviviruses (ISFVs), including two Aedes flavivirus (AeFV) and Yunnan Culex flavivirus strains each, one Culex theileri flavivirus, Yamadai flavivirus (YDFV) and Anopheles-associated flavivirus (AAFV) strains each were detected in Aedes albopictus, Cx. tritaeniorhynchus, Cx. vagans, Cx. pseudovihnui, and An. sinensis pools, respectively. The whole-genome was successfully amplified in one strain of JEV and AeFV each. Phylogenetic analysis using the E gene placed all the newly detected JEV strains into the GI-b genotype. They showed highly nucleotide identities, and were most closely related to the strain detected in Tengchong in 2010. The comparison of the E protein of JEV strains and vaccine-derived strain, showed six amino residue differences. The bias-corrected maximum likelihood estimation values (and 95% confidence interval) for JEV in Cx. tritaeniorhynchus collected in Tengchong in 2018 were 2.4 (1.4–3.9).ConclusionsA potential Japanese encephalitis epidemic focus with the abundance of host mosquitoes and high JEV infection rate was observed in Tengchong. In addition, at least five species of ISFVs co-circulate in this area. This study highlights the importance of widespread and sustained mosquito-based arbovirus surveillance in local areas to prevent the transmission of JEV, and other emerging/re-emerging mosquito-borne pathogens.Graphic

Genetic Heterogeneity among Chicken Infectious Anemia Viruses Detected in Italian Fowl.

Simple SummaryChicken infectious anemia virus (CIAV) is an immunosuppressive pathogen of chickens. In the present study field, vaccine-derived CIAV strains were reported to circulate in different types of chicken flocks. Viruses were successfully obtained from non-invasive samples such as feathers and environmental dust. Genome analysis showed that strains had heterogeneous sequences clustered into different genogroups that possessed genetic markers reported to be correlated with CIAV virulence. This survey contributes to the knowledge of field CIAV distribution maps and increases the existing information available on native isolates around the world.Chicken infectious anemia virus (CIAV) is a pathogen of chickens associated with immunosuppression and with a disease named chicken infectious anemia. The present survey reports an epidemiological study on CIAV distribution in Italian broiler, broiler breeder and backyard chicken flocks. Twenty-five strains were detected by a specifically developed nested PCR protocol, and molecularly characterized by partial VP1 gene or complete genome sequencing. Viral DNA amplification was successfully obtained from non-invasive samples such as feathers and environmental dust. Sequence and phylogenetic analysis showed the circulation of field or potentially vaccine-derived strains with heterogeneous sequences clustered into genogroups II, IIIa, and IIIb. Marker genome positions, reported to be correlated with CIAV virulence, were evaluated in field strains. In conclusion, this is the first survey focused on the molecular characteristics of Italian CIAVs, which have proved to be highly heterogeneous, implementing at the same time a distribution map of field viruses worldwide.

What is new on molecular characteristics of Avian metapneumovirus strains circulating in Europe?

In the present study, one hundred and sixteen partial G gene sequences of Avian metapneumovirus (aMPV) subtype B, obtained during routine diagnostics in different European Countries in the last few years (2014-2019), were analysed by sequence and phylogenetic analyses in order to draw an updated picture of the molecular characteristics of circulating strains. Nucleotide sequences were compared with other sequences of European and non-European aMPV-Bs collected prior to that period or retrieved from GenBank. Phylogenetic relationships among the aMPV-B strains, reconstructed using the maximum likelihood method implemented in MEGA X, demonstrated that aMPV-B has evolved in Europe from its first appearance, frequently displaying a clear relation with the geographic area of detection. The 40% of aMPV-B viruses analysed were classified as vaccine-derived strains, being phylogenetically related, and showing high nucleotide identity with live commercial vaccine strains licensed in Europe. The remaining 60% were classified as field strains since they clustered separately and showed a low nucleotide identity with vaccines and vaccine-derived strains. The phylogenetic tree showed that the virus has continued to evolve from its first appearance in the '80s since more recently detected strains belonged to clades phylogenetically distant from the older strains. Unlike vaccine-derived strains, field strains tended to cluster according to their geographic origin and irrespective of the host species where the viruses had been detected. In conclusion, the molecular characterization of aMPV-B and the differentiation between vaccines and field strains through G gene sequence analysis can be a useful tool towards correct diagnosis and should be routinely applied in order to better address the control strategies.

Human IgA Monoclonal Antibodies That Neutralize Poliovirus, Produced by Hybridomas and Recombinant Expression.

Poliovirus (PV)-specific intestinal IgAs are important for cessation of PV shedding in the gastrointestinal tract following an acute infection with wild type or vaccine-derived PV strains. We sought to produce IgA monoclonal antibodies (mAbs) with PV neutralizing activity. We first performed de novo IgA discovery from primary human B cells using a hybridoma method that allows assessment of mAb binding and expression on the hybridoma surface: On-Cell mAb Screening (OCMS™). Six IgA1 mAbs were cloned by this method; three potently neutralized type 3 Sabin and wt PV strains. The hybridoma mAbs were heterogeneous, expressed in monomeric, dimeric, and aberrant forms. We also used recombinant methods to convert two high-potency anti-PV IgG mAbs into dimeric IgA1 and IgA2 mAbs. Isotype switching did not substantially change their neutralization activities. To purify the recombinant mAbs, Protein L binding was used, and one of the mAbs required a single amino acid substitution in its κ LC in order to enable protein L binding. Lastly, we used OCMS to assess IgA expression on the surface of hybridomas and transiently transfected, adherent cells. These studies have generated potent anti-PV IgA mAbs, for use in animal models, as well as additional tools for the discovery and production of human IgA mAbs.

Vaccine-derived rotavirus strains in infants in England

ObjectiveTo describe infants with acute gastroenteritis symptoms in primary and secondary care who have the Rotarix vaccine-derived G1P[8] rotavirus strain identified in their stools.DesignThis is a prospective national surveillance conducted...

Whole Transcriptome Analyses Reveal Differential mRNA and microRNA Expression Profiles in Primary Human Dermal Fibroblasts Infected with Clinical or Vaccine Strains of Varicella Zoster Virus.

Licensed live attenuated vaccines have been developed to prevent varicella zoster virus (VZV) infection, which causes chickenpox and shingles. The genomic sequences of both clinical- and vaccine-derived VZV strains have been analyzed previously. To further characterize the molecular signatures and complexity of wildtype (clinical) versus attenuated (vaccine-derived) VZV-mediated host cellular responses, we performed high-throughput next generation sequencing to quantify and compare the expression patterns of mRNAs and microRNAs (miRNAs) in primary human dermal fibroblasts (HDFs) infected with wildtype (YC01 low passage) and attenuated (YC01 high passage, SuduVax, and VarilRix) VZV strains. 3D-multidimensional scaling of the differentially expressed genes demonstrated the distinct grouping of wildtype and attenuated strains. In particular, we observed that HDFs infected with attenuated strains had more differentially expressed genes (DEGs) involved in the retinoic-acid inducible gene–I-like receptor and interferon-mediated signaling pathways compared with wildtype strains. Additionally, miRNA expression patterns were profiled following the infection of HDFs with VZV. Small RNA sequencing identified that several miRNAs were upregulated, including miR-146a-5p, which has been associated with other herpesvirus infections, whereas let-7a-3p was downregulated in both wildtype and attenuated VZV-infected cells. This study identified genes and miRNAs that may be essential in VZV pathogenesis.