The Role of the VP4 Attachment Protein in Rotavirus Host Range Restriction in an In Vivo Suckling Mouse Model.

The basis for rotavirus (RV) host range restriction (HRR) is not fully understood but is likely multigenic. RV genes encoding VP3, VP4, NSP1, NSP2, NSP3, and NSP4 have been associated with HRR in various studies. With the exception of NSP1, little is known about the relative contribution of the other RV genes to HRR. VP4 has been linked to HRR because it functions as the RV cell attachment protein, but its actual role in HRR has not been fully assessed. We generated a collection of recombinant RVs (rRVs) in an isogenic murine-like RV genetic background, harboring either heterologous or homologous VP4 genes from simian, bovine, porcine, human, and murine RV strains, and characterized these rRVs in vitro and in vivo. We found that a murine-like rRV encoding a simian VP4 was shed, spread to uninoculated littermates, and induced diarrhea comparably to rRV harboring a murine VP4. However, rRVs carrying VP4s from both bovine and porcine RVs had reduced diarrhea, but no change in fecal shedding was observed. Both diarrhea and shedding were reduced when VP4 originated from a human RV strain. rRVs harboring VP4s from human or bovine RVs did not transmit to uninoculated littermates. We also generated two rRVs harboring reciprocal chimeric murine or bovine VP4. Both chimeras replicated and caused disease as efficiently as the parental strain with a fully murine VP4. These data suggest that the genetic origin of VP4 partially modulates HRR in the suckling mouse and that both the VP8* and VP5* domains independently contribute to pathogenesis and transmission. IMPORTANCE Human group A rotaviruses (RVs) remain the most important cause of severe acute gastroenteritis among infants and young children worldwide despite the introduction of several safe and effective live attenuated vaccines. The lack of knowledge regarding fundamental aspects of RV biology, such as the genetic basis of host range restriction (HRR), has made it difficult to predictively and efficiently design improved, next-generation live attenuated rotavirus vaccines. Here, we engineered a collection of VP4 monoreassortant RVs to systematically explore the role of VP4 in replication, pathogenicity, and spread, as measures of HRR, in a suckling mouse model. The genetic and mechanistic bases of HRR have substantial clinical relevance given that this restriction forms the basis of attenuation for several replication-competent human RV vaccines. In addition, a better understanding of RV pathogenesis and the determinants of RV spread is likely to enhance our ability to improve antiviral drug and therapy development.

[Prevalence of laboratory markers of human respiratory viruses in monkeys of Adler primate center].

INTRODUCTION: The relevance of studying the circulation of human respiratory viruses among laboratory primates is associated with the need to test vaccines and antiviral drugs against these infections on monkeys.The aim of this work was to study the prevalence of serological and molecular markers of human respiratory viral infections in laboratory primates born at the Adler Primate Center and in imported monkeys. MATERIAL AND METHODS: Blood serum samples (n = 1971) and lung autopsy material (n = 26) were obtained from different monkey species. These samples were tested for the presence of serological markers of measles, parainfluenza (PI) types 1, 2, 3, influenza A and B, respiratory syncytial (RS) and adenovirus infections using enzyme immunoassay (ELISA). Detection of RS virus, metapneumovirus, PI virus types 1-4, rhinovirus, coronavirus, and adenoviruses B, C, E and bocavirus nucleic acids in this material was performed by reverse transcription polymerase chain reaction (RT-PCR). RESULTS AND DISCUSSION: The overall prevalence of antibodies (Abs) among all monkeys was low and amounted 11.3% (95% CI: 9.2-13.7%, n = 811) for measles virus, 8.9% (95% CI: 6.2-12.2%, n = 381) for PI type 3 virus, 2.5% (95% CI: 0.8-5.6%, n = 204) for PI type 1 virus, and 7.7% (95% CI: 3.8-13.7%, n = 130) for adenoviruses. When testing 26 autopsy lung samples from monkeys of different species that died from pneumonia, 2 samples from Anubis baboons (Papio аnubis) were positive for of parainfluenza virus type 3 RNA. CONCLUSION: Our data suggest the importance of the strict adherence to the terms of quarantine and mandatory testing of monkey sera for the presence of IgM antibodies to the measles virus that indicate the recent infection. The role of PI virus type 3 in the pathology of the respiratory tract in Anubis baboons has been established.

HERV-Derived Ervpb1 Is Conserved in Simiiformes, Exhibiting Expression in Hematopoietic Cell Lineages Including Macrophages.

(1) Background: The ERVPb1 gene in humans is derived from an envelope (Env) gene of a human endogenous retrovirus group, HERV-P(b). The ERVPb1 gene reportedly has a conserved open reading frame (ORF) in Old World monkeys. Although its forced expression led to cell-fusion in an ex vivo cell culture system, like other Env-derived genes such as syncytin-1 and -2, its mRNA expression is not placenta-specific, but almost ubiquitous, albeit being quite low in human tissues and organs, implying a distinct role for ERVPb1. (2) Methods: To elucidate the cell lineage(s) in which the ERVPb1 protein is translated in human development, we developed a novel, highly sensitive system for detecting HERV-derived proteins/peptides expressed in the tissue differentiation process of human induced pluripotent stem cells (iPSCs). (3) Results: We first determined that ERVPb1 is also conserved in New World monkeys. Then, we showed that the ERVPb1 protein is translated from a uniquely spliced ERVPb1 transcript in hematopoietic cell lineages, including a subset of macrophages, and further showed that its mRNA expression is upregulated by lipopolysaccharide (LPS) stimulation in primary human monocytes. (4) Conclusions: ERVPb1 is unique to Simiiformes and actually translated in hematopoietic cell lineages, including a subset of macrophages.

IP-10 and CXCR3 signaling inhibit Zika virus replication in human prostate cells.

Our previous studies have shown that Zika virus (ZIKV) replicates in human prostate cells, suggesting that the prostate may serve as a long-term reservoir for virus transmission. Here, we demonstrated that the innate immune responses generated to three distinct ZIKV strains (all isolated from human serum) were significantly different and dependent on their passage history (in mosquito, monkey, or human cells). In addition, some of these phenotypic differences were reduced by a single additional cell culture passage, suggesting that viruses that have been passaged more than 3 times from the patient sample will no longer reflect natural phenotypes. Two of the ZIKV strains analyzed induced high levels of the IP-10 chemokine and IFNγ in human prostate epithelial and stromal mesenchymal stem cells. To further understand the importance of these innate responses on ZIKV replication, we measured the effects of IP-10 and its downstream receptor, CXCR3, on RNA and virus production in prostate cells. Treatment with IP-10, CXCR3 agonist, or CXCR3 antagonist significantly altered ZIKV viral gene expression, depending on their passage in cells of relevant hosts (mosquito or human). We detected differences in gene expression of two primary CXCR3 isoforms (CXCR3-A and CXCR3-B) on the two cell types, possibly explaining differences in viral output. Lastly, we examined the effects of IP-10, agonist, or antagonist on cell death and proliferation under physiologically relevant infection rates, and detected no significant differences. Although we did not measure protein expression directly, our results indicate that CXCR3 signaling may be a target for therapeutics, to ultimately stop sexual transmission of this virus.

Natural killer cell responses to emerging viruses of zoonotic origin.

Emerging viral diseases pose a major threat to public health worldwide. Nearly all emerging viruses, including Ebola, Dengue, Nipah, West Nile, Zika, and coronaviruses (including SARS-Cov2, the causative agent of the current COVID-19 pandemic), have zoonotic origins, indicating that animal-to-human transmission constitutes a primary mode of acquisition of novel infectious diseases. Why these viruses can cause profound pathologies in humans, while natural reservoir hosts often show little evidence of disease is not completely understood. Differences in the host immune response, especially within the innate compartment, have been suggested to be involved in this divergence. Natural killer (NK) cells are innate lymphocytes that play a critical role in the early antiviral response, secreting effector cytokines and clearing infected cells. In this review, we will discuss the mechanisms through which NK cells interact with viruses, their contribution towards maintaining equilibrium between the virus and its natural host, and their role in disease progression in humans and other non-natural hosts.

Generation of Simian Rotavirus Reassortants with VP4- and VP7-Encoding Genome Segments from Human Strains Circulating in Africa Using Reverse Genetics.

Human rotavirus A (RVA) causes acute gastroenteritis in infants and young children. The broad use of two vaccines, which are based on RVA strains from Europe and North America, significantly reduced rotavirus disease burden worldwide. However, a lower vaccine effectiveness is recorded in some regions of the world, such as sub-Saharan Africa, where diverse RVA strains are circulating. Here, a plasmid-based reverse genetics system was used to generate simian RVA reassortants with VP4 and VP7 proteins derived from African human RVA strains not previously adapted to cell culture. We were able to rescue 1/3 VP4 mono-reassortants, 3/3 VP7 mono-reassortants, but no VP4/VP7 double reassortant. Electron microscopy showed typical triple-layered virus particles for the rescued reassortants. All reassortants stably replicated in MA-104 cells; however, the VP4 reassortant showed significantly slower growth compared to the simian RVA or the VP7 reassortants. The results indicate that, at least in cell culture, human VP7 has a high reassortment potential, while reassortment of human VP4 from unadapted human RVA strains with simian RVA seems to be limited. The characterized reassortants may be useful for future studies investigating replication and reassortment requirements of rotaviruses as well as for the development of next generation rotavirus vaccines.

Phylogeography of Kyasanur Forest Disease virus in India (1957-2017) reveals evolution and spread in the Western Ghats region.

The Kyasanur Forest Disease (KFD) has become a major public health problem in the State of Karnataka, India where the disease was first identified and in Tamil Nadu, Maharashtra, Kerala, and Goa covering the Western Ghats region of India. The incidence of positive cases and distribution of the Kyasanur Forest Disease virus (KFDV) in different geographical regions raises the need to understand the evolution and spatiotemporal transmission dynamics. Phylogeography analysis based on 48 whole genomes (46 from this study) and additionally 28 E-gene sequences of KFDV isolated from different regions spanning the period 1957-2017 was thus undertaken. The mean evolutionary rates based the E-gene was marginally higher than that based on the whole genomes. A subgroup of KFDV strains (2006-2017) differing from the early Karnataka strains (1957-1972) by ~2.76% in their whole genomes and representing spread to different geographical areas diverged around 1980. Dispersal from Karnataka to Goa and Maharashtra was indicated. Maharashtra represented a new source for transmission of KFDV since ~2013. Significant evidence of adaptive evolution at site 123 A/T located in the vicinity of the envelope protein dimer interface may have functional implications. The findings indicate the need to curtail the spread of KFDV by surveillance measures and improved vaccination strategies.

Bocaparvovirus, Erythroparvovirus and Tetraparvovirus in New World Primates from Central America.

Parvoviruses in the genera Bocaparvovirus (HBoV), Erythroparvovirus (B19) and Tetraparvovirus (PARV4) are the only autonomous parvoviruses known to be associated with human and non-human primates based on studies and clinical cases in humans worldwide and non-human primates in Asia and Africa. Here, the presence of these agents with pathogenic potential was assessed by PCR in blood and faeces from 55 howler monkeys, 112 white-face monkeys, 3 squirrel monkeys and 127 spider monkeys in Costa Rica and El Salvador. Overall, 3.7% (11/297) of the monkeys had HboV DNA, 0.67% (2/297) had B19 DNA, and 14.1% (42/297) had PARV4 DNA, representing the first detection of these viruses in New World Primates (NWP). Sex was significantly associated with the presence of HBoV, males having greater risk up to nine times compared with females. Captivity was associated with increased prevalence for PARV4 and when all viruses were analysed together. This study provides compelling molecular evidence of parvoviruses in NWPs and underscores the importance of future research aimed at understanding how these viruses behave in natural environments of the Neotropics and what variables may favour their presence and transmission.

Epizootics due to Yellow Fever Virus in São Paulo State, Brazil: viral dissemination to new areas (2016-2017).

Beginning in late 2016 Brazil faced the worst outbreak of Yellow Fever in recent decades, mainly located in southeastern rural regions of the country. In the present study we characterize the Yellow Fever Virus (YFV) associated with this outbreak in São Paulo State, Brazil. Blood or tissues collected from 430 dead monkeys and 1030 pools containing a total of 5,518 mosquitoes were tested for YFV by quantitative RT-PCR, immunohistochemistry (IHC) and indirect immunofluorescence. A total of 67 monkeys were YFV-positive and 3 pools yielded YFV following culture in a C6/36 cell line. Analysis of five nearly full length genomes of YFV from collected samples was consistent with evidence that the virus associated with the São Paulo outbreak originated in Minas Gerais. The phylogenetic analysis also showed that strains involved in the 2016-2017 outbreak in distinct Brazilian states (i.e., Minas Gerais, Rio de Janeiro, Espirito Santo) intermingled in maximum-likelihood and Bayesian trees. Conversely, the strains detected in São Paulo formed a monophyletic cluster, suggesting that they were local-adapted. The finding of YFV by RT-PCR in five Callithrix monkeys who were all YFV-negative by histopathology or immunohistochemistry suggests that this YFV lineage circulating in Sao Paulo is associated with different outcomes in Callithrix when compared to other monkeys.

Kyasanur Forest Disease in India: innovative options for intervention.

Kyasanur Forest Disease (KFD) is a tick-borne hemorrhagic fever of human, caused by Kyasanur forest disease virus (KFDV) in India. The tick, Haemaphysalis spinigera, has been incriminated as the vector of KFDV. In human, KFD clinically presents with high fever, frontal headache, and severe myalgia, followed by bleeding from the nasal cavity, throat, gingivae, and in some cases, gastrointestinal tract. The mortality rate in KFDV infected cases is estimated to be 3-10%. Monkeys infected with the virus also develop the disease and die. Though the incidence of KFD was found to be confined only to the sylvatic area of Shimoga district in Karnataka state in India during 1967, recent reports indicate its expanding potential to the neighboring states such as Kerala, Tamil Nadu, and Goa. The administration of an indigenous, inactivated tissue culture vaccine was found to drastically decrease the percentage of incidence; however, the recurrence of KFD in vaccinated subjects warrants innovative strategies for effective control of the infection. The present communication proposes and discusses innovative intervention strategies for the effective prevention and control of KFD in India.

Seroprevalence for norovirus genogroups GII and GIV in captive non-human primates.

Noroviruses (NoVs) are a major cause of epidemic gastroenteritis in children and adults. Several pieces of evidence suggest that viruses genetically and antigenically closely related to human NoVs might infect animals, raising public health concerns about potential cross-species transmission. The natural susceptibility of non-human primates (NPHs) to human NoV infections has already been reported, but a limited amount of data is currently available. In order to start filling this gap, we screened a total of 86 serum samples of seven different species of NPHs housed at the Zoological Garden (Bioparco) of Rome (Italy), collected between 2001 and 2017, using an enzyme-linked immunosorbent assay (ELISA) based on virus-like particles (VLPs) of human GII.4 and GIV.1 NoVs. Antibodies specific for both genotypes were detected with an overall prevalence of 32.6%. In detail, IgG antibodies against GII.4 NoVs were found in 18 Japanese macaques (29.0%, 18/62), a mandrill (10.0%, 1/10), a white-crowned mangabey (16.6%, 1/6) and in an orangutan (33.3%, 1/3). Twelve macaques (19.3%, 12/62), five mandrills (50.0%, 5/10), two chimpanzees (100%, 2/2) and a white-crowned mangabey (16.6%, 1/6) showed antibodies for GIV.1 NoVs. The findings of this study confirm the natural susceptibility of captive NHPs to GII NoV infections. In addition, IgG antibodies against GIV.1 were detected, suggesting that NHPs are exposed to GIV NoVs or to antigenically related NoV strains.

Key Viral Adaptations Preceding the AIDS Pandemic.

HIV, the causative agent of AIDS, has a complex evolutionary history involving several cross-species transmissions and recombination events as well as changes in the repertoire and function of its accessory genes. Understanding these events and the adaptations to new host species provides key insights into innate defense mechanisms, viral dependencies on cellular factors, and prerequisites for the emergence of the AIDS pandemic. In addition, understanding the factors and adaptations required for the spread of HIV in the human population helps to better assess the risk of future lentiviral zoonoses and provides clues to how improved control of viral replication can be achieved. Here, we summarize our current knowledge on viral features and adaptations preceding the AIDS pandemic. We aim at providing a viral point of view, focusing on known key hurdles of each cross-species transmission and the mechanisms that HIV and its simian precursors evolved to overcome them.

Predicting Yellow Fever Through Species Distribution Modeling of Virus, Vector, and Monkeys.

Mapping yellow fever (YF) risk is often based on place of infection of human cases, whereas the circulation between nonhuman primates (NHP) and vectors is neglected. In 2008/2009, YF devastated NHP at the southern limit of the disease in the Americas. In view of the recent expansion of YF in Brazil, we modeled the environmental suitability for YF with data from 2008/2009 epizootic, the distribution of NHP (Alouatta spp.), and the mosquito (Haemagogus leucocelaenus) using the maximum entropy algorithm (Maxent) to define risk areas for YF and their main environmental predictors. We evaluated points of occurrence of YF based on dates of confirmed deaths of NHP in three periods, from October 2008 to: December 2008, March 2009, and June 2009. Variables with greatest influence on suitability for YF were seasonality in water vapor pressure (36%), distribution of NHP (32%), maximum wind speed (11%), annual mean rainfall (7%), and maximum temperature in the warmest month (5%). Models of early periods of the epizootic identified suitability for YF in localities that recorded NHP deaths only months later, demonstrating usefulness of the approach for predicting the disease spread. Our data supported influence of rainfall, air humidity, and ambient temperature on the distribution of epizootics. Wind was highlighted as a predicting variable, probably due to its influence on the dispersal of vectors infected with YF in fragmented landscapes. Further studies on the role of wind are necessary to improve our understanding of the occurrence of YF and other arboviruses and their dispersal in the landscape.

Mass spectrometry-based identification and whole-genome characterisation of the first pteropine orthoreovirus isolated from monkey faeces in Thailand.

BACKGROUND: The pteropine orthoreovirus (PRV) was isolated from monkey (Macaca fascicularis) faecal samples collected from human-inhabited areas in Lopburi Province, Thailand. These samples were initially obtained to survey for the presence of hepatitis E virus (HEV). RESULTS: Two virus isolates were retrieved by virus culture of 55 monkey faecal samples. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was successfully used to identify the viruses as the segmented dsRNA orthoreovirus. Phylogenetic analysis of the Lopburi orthoreovirus whole-genomes revealed relationships with the well-characterised PRVs Pulau (segment L1), Cangyuan (segments L2, M3 and S3), Melaka (segments L3 and M2), Kampar (segments M1 and S2) and Sikamat (segments S1 and S4) of Southeast Asia and China with nucleotide sequence identities of 93.5-98.9%. RT-PCR showed that PRV was detected in 10.9% (6/55) and HEV was detected in 25.5% (14/55) of the monkey faecal samples. CONCLUSIONS: PRV was isolated from monkey faeces for the first time in Thailand via viral culture and LC-MS/MS. The genetic diversity of the virus genome segments suggested a re-assortment within the PRV species group. The overall findings emphasise that monkey faeces can be sources of zoonotic viruses, including PRV and HEV, and suggest the need for active virus surveillance in areas of human and monkey co-habitation to prevent and control emerging zoonotic diseases in the future.

Monkey Models and HIV Vaccine Research.

Since the discovery of acquired immunodeficiency syndrome (AIDS) in 1981, it has been extremely difficult to develop an effective vaccine or a therapeutic cure despite over 36 years of global efforts. One of the major reasons is due to the lack of an immune-competent animal model that supports live human immunodeficiency virus (HIV) infection and disease progression such that vaccine-induced correlates of protection and efficacy can be determined clearly before human trials. Nevertheless, rhesus macaques infected with simian immunodeficiency virus (SIV) and chimeric simian human immunodeficiency virus (SHIV) have served as invaluable models not only for understanding AIDS pathogenesis but also for studying HIV vaccine and cure. In this chapter, therefore, we summarize major scientific evidence generated in these models since the beginning of the AIDS pandemic. Hopefully, the accumulated knowledge and lessons contributed by thousands of scientists will be useful in promoting the search of an ultimate solution to end HIV/AIDS.

Using barcoded Zika virus to assess virus population structure in vitro and in Aedes aegypti mosquitoes.

Arboviruses such as Zika virus (ZIKV, Flaviviridae; Flavivirus) must replicate in both mammalian and insect hosts possessing strong immune defenses. Accordingly, transmission between and replication within hosts involves genetic bottlenecks, during which viral population size and genetic diversity may be significantly reduced. To help quantify these bottlenecks and their effects, we constructed 4 "barcoded" ZIKV populations that theoretically contain thousands of barcodes each. After identifying the most diverse barcoded virus, we passaged this virus 3 times in 2 mammalian and mosquito cell lines and characterized the population using deep sequencing of the barcoded region of the genome. C6/36 maintain higher barcode diversity, even after 3 passages, than Vero. Additionally, field-caught mosquitoes exposed to the virus to assess bottlenecks in a natural host. A progressive reduction in barcode diversity occurred throughout systemic infection of these mosquitoes. Differences in bottlenecks during systemic spread were observed between different populations of Aedes aegypti.

Outbreak of Yellow Fever among Nonhuman Primates, Espirito Santo, Brazil, 2017.

In January 2017, a yellow fever outbreak occurred in Espirito Santo, Brazil, where human immunization coverage is low. Histologic, immunohistologic, and PCR examinations were performed for 22 deceased nonhuman New World primates; typical yellow fever features were found in 21. Diagnosis in nonhuman primates prompted early public health response.

Screening for Viral Pathogens in African Simian Bushmeat Seized at A French Airport.

Illegal bushmeat traffic is an important threat to biodiversity conservation of several endangered species and may contribute to the emergence and spread of infectious diseases in humans. The hunting, manipulation and consumption of wildlife-based products, especially those of primate origin, may be a threat to human health; however, few studies have investigated the role of bushmeat trade and consumption as a potential source of human infections to date. In this study, we report the screening of viral pathogens in African simian game seized by French customs at Toulouse Blagnac Airport. Epifluorescence microscopy revealed the presence of virus-like particles in the samples, and further metagenomic sequencing of the DNA and RNA viromes confirmed the presence of sequences related to the Siphoviridae, Myoviridae and Podoviridae bacteriophage families; some of them infecting bacterial hosts that could be potentially pathogenic for humans. To increase the sensitivity of detection, twelve pan-generic PCRs targeting several viral zoonoses were performed, but no positive signal was detected. A large-scale inventory of bacteria, viruses and parasites is urgently needed to globally assess the risk for human health of the trade, manipulation and consumption of wildlife-related bushmeat.