Taxonomy of the order Bunyavirales: update 2019.

In February 2019, following the annual taxon ratification vote, the order Bunyavirales was amended by creation of two new families, four new subfamilies, 11 new genera and 77 new species, merging of two species, and deletion of one species. This article presents the updated taxonomy of the order Bunyavirales now accepted by the International Committee on Taxonomy of Viruses (ICTV).

Taxonomy of the order Bunyavirales: second update 2018.

In October 2018, the order Bunyavirales was amended by inclusion of the family Arenaviridae, abolishment of three families, creation of three new families, 19 new genera, and 14 new species, and renaming of three genera and 22 species. This article presents the updated taxonomy of the order Bunyavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

Enhancement of Microbiota in Healthy Macaques Results in Beneficial Modulation of Mucosal and Systemic Immune Function.

Given the critical role of mucosal surfaces in susceptibility to infection, it is imperative that effective mucosal responses are induced when developing efficacious vaccines and prevention strategies for infection. Modulating the microbiota in the gastrointestinal (GI) tract through the use of probiotics (PBio) is a safe and well-tolerated approach to enhance mucosal and overall health. We assessed the longitudinal impact of daily treatment with the VSL#3 probiotic on cellular and humoral immunity and inflammation in healthy macaques. PBio therapy resulted in significantly increased frequencies of B cells expressing IgA in the colon and lymph node (LN), likely because of significantly increased LN T follicular helper cell frequencies and LN follicles. Increased frequencies of IL-23(+) APCs in the colon were found post-PBio treatment, which correlated with LN T follicular helper cells. Finally, VSL#3 significantly downmodulated the response of TLR2-, TLR3-, TLR4-, and TLR9-expressing HEK293 cells to stimulation with Pam3CSK4, polyinosinic-polycytidylic acid, LPS, and ODN2006, respectively. These data provide a mechanism for the beneficial impact of PBio on mucosal health and implicates the use of PBio therapy in the context of vaccination or preventative approaches to enhance protection from mucosal infection by improving immune defenses at the mucosal portal of entry.

Early nasopharyngeal microbial signature associated with severe influenza in children: a retrospective pilot study.

A few studies have highlighted the importance of the respiratory microbiome in modulating the frequency and outcome of viral respiratory infections. However, there are insufficient data on the use of microbial signatures as prognostic biomarkers to predict respiratory disease outcomes. In this study, we aimed to evaluate whether specific bacterial community compositions in the nasopharynx of children at the time of hospitalization are associated with different influenza clinical outcomes. We utilized retrospective nasopharyngeal (NP) samples (n=36) collected at the time of hospital arrival from children who were infected with influenza virus and had been symptomatic for less than 2 days. Based on their clinical course, children were classified into two groups: patients with mild influenza, and patients with severe respiratory or neurological complications. We implemented custom 16S rRNA gene sequencing, metagenomic sequencing and computational analysis workflows to classify the bacteria present in NP specimens at the species level. We found that increased bacterial diversity in the nasopharynx of children was strongly associated with influenza severity. In addition, patients with severe influenza had decreased relative abundance of Staphylococcus aureus and increased abundance of Prevotella (including P. melaninogenica), Streptobacillus, Porphyromonas, Granulicatella (including G. elegans), Veillonella (including V. dispar), Fusobacterium and Haemophilus in their nasopharynx. This pilot study provides proof-of-concept data for the use of microbial signatures as prognostic biomarkers of influenza outcomes. Further large prospective cohort studies are needed to refine and validate the performance of such microbial signatures in clinical settings.

Effects of Fecal Microbial Transplantation on Microbiome and Immunity in Simian Immunodeficiency Virus-Infected Macaques.

UNLABELLED: An altered intestinal microbiome during chronic human immunodeficiency virus (HIV) infection is associated with mucosal dysfunction, inflammation, and disease progression. We performed a preclinical evaluation of the safety and efficacy of fecal microbiota transplantation (FMT) as a potential therapeutic in HIV-infected individuals. Antiretroviral-treated, chronically simian immunodeficiency virus (SIV)-infected rhesus macaques received antibiotics followed by FMT. The greatest microbiota shift was observed after antibiotic treatment. The bacterial community composition at 2 weeks post-FMT resembled the pre-FMT community structure, although differences in the abundances of minor bacterial populations remained. Immunologically, we observed significant increases in the number of peripheral Th17 and Th22 cells and reduced CD4(+) T cell activation in gastrointestinal tissues post-FMT. Importantly, the transplant was well tolerated with no negative clinical side effects. Although this pilot study did not control for the differential contributions of antibiotic treatment and FMT to the observed results, the data suggest that FMT may have beneficial effects that should be further evaluated in larger studies. IMPORTANCE: Due to the immunodeficiency and chronic inflammation that occurs during HIV infection, determination of the safety of FMT is crucial to prevent deleterious consequences if it is to be used as a treatment in the future. Here we used the macaque model of HIV infection and performed FMT on six chronically SIV-infected rhesus macaques on antiretroviral treatment. In addition to providing a preclinical demonstration of the safety of FMT in primates infected with a lentivirus, this study provided a unique opportunity to examine the relationships between alterations to the microbiome and immunological parameters. In this study, we found increased numbers of Th17 and Th22 cells as well as decreased activation of CD4(+) T cells post-FMT, and these changes correlated most strongly across all sampling time points with lower-abundance taxonomic groups and other taxonomic groups in the colon. Overall, these data provide evidence that changes in the microbiome, particularly in terms of diversity and changes in minor populations, can enhance immunity and do not have adverse consequences.

A single positively selected West Nile viral mutation confers increased virogenesis in American crows.

West Nile virus (WNV), first recognized in North America in 1999, has been responsible for the largest arboviral epiornitic and epidemic of human encephalitis in recorded history. Despite the well-described epidemiological patterns of WNV in North America, the basis for the emergence of WNV-associated avian pathology, particularly in the American crow (AMCR) sentinel species, and the large scale of the North American epidemic and epiornitic is uncertain. We report here that the introduction of a T249P amino acid substitution in the NS3 helicase (found in North American WNV) in a low-virulence strain was sufficient to generate a phenotype highly virulent to AMCRs. Furthermore, comparative sequence analyses of full-length WNV genomes demonstrated that the same site (NS3-249) was subject to adaptive evolution. These phenotypic and evolutionary results provide compelling evidence for the positive selection of a mutation encoding increased viremia potential and virulence in the AMCR sentinel bird species.

Nhumirim virus, a novel flavivirus isolated from mosquitoes from the Pantanal, Brazil.

We describe the isolation of a novel flavivirus, isolated from a pool of mosquitoes identified as Culex (Culex) chidesteri collected in 2010 in the Pantanal region of west-central Brazil. The virus is herein designated Nhumirim virus (NHUV) after the name of the ranch from which the mosquito pool was collected. Flavivirus RNA was detected by real-time RT-PCR of homogenized mosquitoes and from the corresponding C6/36 culture supernatant. Based on full-genome sequencing, the virus isolate was genetically distinct from but most closely related to Barkedji virus (BJV), a newly described flavivirus from Senegal. Phylogenetic analysis demonstrated that NHUV grouped with mosquito-borne flaviviruses forming a clade with BJV. This clade may be genetically intermediate between the Culex-borne flaviviruses amplified by birds and the insect-only flaviviruses.

Characterization of a novel insect-specific flavivirus from Brazil: potential for inhibition of infection of arthropod cells with medically important flaviviruses.

In the past decade, there has been an upsurge in the number of newly described insect-specific flaviviruses isolated pan-globally. We recently described the isolation of a novel flavivirus (tentatively designated 'Nhumirim virus'; NHUV) that represents an example of a unique subset of apparently insect-specific viruses that phylogenetically affiliate with dual-host mosquito-borne flaviviruses despite appearing to be limited to replication in mosquito cells. We characterized the in vitro growth potential and 3' untranslated region (UTR) sequence homology with alternative flaviviruses, and evaluated the virus's capacity to suppress replication of representative Culex spp.-vectored pathogenic flaviviruses in mosquito cells. Only mosquito cell lines were found to support NHUV replication, further reinforcing the insect-specific phenotype of this virus. Analysis of the sequence and predicted RNA secondary structures of the 3' UTR indicated NHUV to be most similar to viruses within the yellow fever serogroup and Japanese encephalitis serogroup, and viruses in the tick-borne flavivirus clade. NHUV was found to share the fewest conserved sequence elements when compared with traditional insect-specific flaviviruses. This suggests that, despite apparently being insect specific, this virus probably diverged from an ancestral mosquito-borne flavivirus. Co-infection experiments indicated that prior or concurrent infection of mosquito cells with NHUV resulted in a significant reduction in virus production of West Nile virus (WNV), St Louis encephalitis virus (SLEV) and Japanese encephalitis virus. The inhibitory effect was most effective against WNV and SLEV with over a 10(6)-fold and 10(4)-fold reduction in peak titres, respectively.

West Nile virus and non-West Nile virus mortality and coinfection of American crows (Corvus brachyrhynchos) in California.

American crows are acutely sensitive to West Nile virus (WNV) infection, and crow mortality has been used in WNV surveillance to monitor enzootic transmission. However, non-WNV sources of mortality could reduce the reliability of crow death as a surveillance tool. Here, using a combination of histopathologic, toxicologic, virologic, and molecular techniques we describe causes of mortality in 67 American crows (Corvus brachyrhynchos) that were collected from a population in the Sacramento Valley of California in 2012 and 2013. Evidence of infectious disease was detected in 70% (47/67) of carcasses. The majority of deaths were linked to a suite of non-WNV viral, bacterial, and fungal infections (39%; 23/59 cases), WNV (36%; 24/67 cases), and an acute toxic event (25%; 15/59 cases). Coinfections were detected in 20% (12/59) of birds and frequently were associated with WNV and poxviral dermatitis. Inferences about WNV activity based on crow mortality should be supported by laboratory confirmation because crow mortality frequently can be caused by other infectious diseases or toxic events.

Enriching pathogen transcripts from infected samples: a capture-based approach to enhanced host-pathogen RNA sequencing.

To fully understand the interactions of a pathogen with its host, it is necessary to analyze the RNA transcripts of both the host and pathogen throughout the course of an infection. Although this can be accomplished relatively easily on the host side, the analysis of pathogen transcripts is complicated by the overwhelming amount of host RNA isolated from an infected sample. Even with the read depth provided by second-generation sequencing, it is extremely difficult to get enough pathogen reads for an effective gene-level analysis. In this study, we describe a novel capture-based technique and device that considerably enriches for pathogen transcripts from infected samples. This versatile method can, in principle, enrich for any pathogen in any infected sample. To test the technique's efficacy, we performed time course tissue culture infections using Rift Valley fever virus and Francisella tularensis. At each time point, RNA sequencing (RNA-Seq) was performed and the results of the treated samples were compared with untreated controls. The capture of pathogen transcripts, in all cases, led to more than an order of magnitude enrichment of pathogen reads, greatly increasing the number of genes hit, the coverage of those genes, and the depth at which each transcript was sequenced.

Peregrine: A rapid and unbiased method to produce strand-specific RNA-Seq libraries from small quantities of starting material.

Use of second generation sequencing (SGS) technologies for transcriptional profiling (RNA-Seq) has revolutionized transcriptomics, enabling measurement of RNA abundances with unprecedented specificity and sensitivity and the discovery of novel RNA species. Preparation of RNA-Seq libraries requires conversion of the RNA starting material into cDNA flanked by platform-specific adaptor sequences. Each of the published methods and commercial kits currently available for RNA-Seq library preparation suffers from at least one major drawback, including long processing times, large starting material requirements, uneven coverage, loss of strand information and high cost. We report the development of a new RNA-Seq library preparation technique that produces representative, strand-specific RNA-Seq libraries from small amounts of starting material in a fast, simple and cost-effective manner. Additionally, we have developed a new quantitative PCR-based assay for precisely determining the number of PCR cycles to perform for optimal enrichment of the final library, a key step in all SGS library preparation workflows.

Structural gene (prME) chimeras of St Louis encephalitis virus and West Nile virus exhibit altered in vitro cytopathic and growth phenotypes.

Despite utilizing the same avian hosts and mosquito vectors, St Louis encephalitis virus (SLEV) and West Nile virus (WNV) display dissimilar vector-infectivity and vertebrate-pathogenic phenotypes. SLEV exhibits a low oral infection threshold for Culex mosquito vectors and is avirulent in avian hosts, producing low-magnitude viraemias. In contrast, WNV is less orally infective to mosquitoes and elicits high-magnitude viraemias in a wide range of avian species. In order to identify the genetic determinants of these different phenotypes and to assess the utility of mosquito and vertebrate cell lines for recapitulating in vivo differences observed between these viruses, reciprocal WNV and SLEV pre-membrane and envelope protein (prME) chimeric viruses were generated and growth of these mutant viruses was characterized in mammalian (Vero), avian (duck) and mosquito [Aedes (C6/36) and Culex (CT)] cells. In both vertebrate lines, WNV grew to 100-fold higher titres than SLEV, and growth and cytopathogenicity phenotypes, determined by chimeric phenotypes, were modulated by genetic elements outside the prME gene region. Both chimeras exhibited distinctive growth patterns from those of SLEV in C6/36 cells, indicating the role of both structural and non-structural gene regions for growth in this cell line. In contrast, growth of chimeric viruses was indistinguishable from that of virus containing homologous prME genes in CT cells, indicating that structural genetic elements could specifically dictate growth differences of these viruses in relevant vectors. These data provide genetic insight into divergent enzootic maintenance strategies that could also be useful for the assessment of emergence mechanisms of closely related flaviviruses.

Envelope and pre-membrane protein structural amino acid mutations mediate diminished avian growth and virulence of a Mexican West Nile virus isolate.

The hallmark attribute of North American West Nile virus (WNV) strains has been high pathogenicity in certain bird species. Surprisingly, this avian virulent WNV phenotype has not been observed during its geographical expansion into the Caribbean, Central America and South America. One WNV variant (TM171-03-pp1) isolated in Mexico has demonstrated an attenuated phenotype in two widely distributed North American bird species, American crows (AMCRs) and house sparrows (HOSPs). In order to identify genetic determinants associated with attenuated avian replication of the TM171-03-pp1 variant, chimeric viruses between the NY99 and Mexican strains were generated, and their replicative capacity was assessed in cell culture and in AMCR, HOSP and house finch avian hosts. The results demonstrated that mutations in both the pre-membrane (prM-I141T) and envelope (E-S156P) genes mediated the attenuation phenotype of the WNV TM171-03-pp1 variant in a chicken macrophage cell line and in all three avian species assayed. Inclusion of the prM-I141T and E-S156P TM171-03-pp1 mutations in the NY99 backbone was necessary to achieve the avian attenuation level of the Mexican virus. Furthermore, reciprocal incorporation of both prM-T141I and E-P156S substitutions into the Mexican virus genome was necessary to generate a virus that exhibited avian virulence equivalent to the NY99 virus. These structural changes may indicate the presence of new evolutionary pressures exerted on WNV populations circulating in Latin America or may signify a genetic bottleneck that has constrained their epiornitic potential in alternative geographical locations.

Stone Lakes virus (family Togaviridae, genus Alphavirus), a variant of Fort Morgan virus isolated from swallow bugs (Hemiptera: Cimicidae) west of the Continental Divide.

Multiple isolates of an alphaviruses within the western equine encephalomyelitis-serocomplex that were related closely to Ft. Morgan and its variant Buggy Creek virus were made from swallow bugs, Oeciacus vicarius Horvath (Hemiptera: Cimicidae), collected from cliff swallow (Petrochelidon pyrrhonota) nests at the Stone Lakes National Wildlife Refuge, Sacramento County, CA, during the summers of 2005 and 2006. This virus (hereafter Stone Lakes virus, family Togaviridae, genus Alphavirus, STLV) was the first record of this viral group west of the Continental Divide. STLV replicated well in Vero and other vertebrate cell cultures but failed to replicate in C6/36 cells or infect Culex tarsalis Coquillett mosquitoes. STLV failed to produce elevated viremias in adult chickens or house sparrows and was weakly immunogenic. In addition, STLV was not isolated from cliff swallow nestlings nor was antibody detected in adults collected at mist nets. We suggest that STL and related swallow bug viruses may be primarily infections of cimicids that are maintained and amplified either by vertical or nonviremic transmission and that cliff swallows may primarily be important as a bloodmeal source for the bugs rather than as an amplification host for the viruses.

N-linked glycosylation of the West Nile virus envelope protein is not a requisite for avian virulence or vector competence.

The N-linked glycosylation motif at amino acid position 154-156 of the envelope (E) protein of West Nile virus (WNV) is linked to enhanced murine neuroinvasiveness, avian pathogenicity and vector competence. Naturally occurring isolates with altered E protein glycosylation patterns have been observed in WNV isolates; however, the specific effects of these polymorphisms on avian host pathogenesis and vector competence have not been investigated before. In the present study, amino acid polymorphisms, NYT, NYP, NYF, SYP, SYS, KYS and deletion (A'DEL), were reverse engineered into a parental WNV (NYS) cDNA infectious clone to generate WNV glycosylation mutant viruses. These WNV glycosylation mutant viruses were characterized for in vitro growth, pH-sensitivity, temperature-sensitivity and host competence in American crows (AMCR), house sparrows (HOSP) and Culex quinquefasciatus. The NYS and NYT glycosylated viruses showed higher viral replication, and lower pH and temperature sensitivity than NYP, NYF, SYP, SYS, KYS and A'DEL viruses in vitro. Interestingly, in vivo results demonstrated asymmetric effects in avian and mosquito competence that were independent of the E-protein glycosylation status. In AMCRs and HOSPs, all viruses showed comparable viremias with the exception of NYP and KYS viruses that showed attenuated phenotypes. Only NYP showed reduced vector competence in both Cx. quinquefasciatus and Cx. tarsalis. Glycosylated NYT exhibited similar avian virulence properties as NYS, but resulted in higher mosquito oral infectivity than glycosylated NYS and nonglycosylated, NYP, NYF, SYP and KYS mutants. These data demonstrated that amino acid polymorphisms at E154/156 dictate differential avian host and vector competence phenotypes independent of E-protein glycosylation status.

West Nile and St. Louis encephalitis viral genetic determinants of avian host competence.

West Nile virus (WNV) and St. Louis encephalitis (SLEV) virus are enzootically maintained in North America in cycles involving the same mosquito vectors and similar avian hosts. However, these viruses exhibit dissimilar viremia and virulence phenotypes in birds: WNV is associated with high magnitude viremias that can result in mortality in certain species such as American crows (AMCRs, Corvus brachyrhynchos) whereas SLEV infection yields lower viremias that have not been associated with avian mortality. Cross-neutralization of these viruses in avian sera has been proposed to explain the reduced circulation of SLEV since the introduction of WNV in North America; however, in 2015, both viruses were the etiologic agents of concurrent human encephalitis outbreaks in Arizona, indicating the need to re-evaluate host factors and cross-neutralization responses as factors potentially affecting viral co-circulation. Reciprocal chimeric WNV and SLEV viruses were constructed by interchanging the pre-membrane (prM)-envelope (E) genes, and viruses subsequently generated were utilized herein for the inoculation of three different avian species: house sparrows (HOSPs; Passer domesticus), house finches (Haemorhous mexicanus) and AMCRs. Cross-protective immunity between parental and chimeric viruses were also assessed in HOSPs. Results indicated that the prM-E genes did not modulate avian replication or virulence differences between WNV and SLEV in any of the three avian species. However, WNV-prME proteins did dictate cross-protective immunity between these antigenically heterologous viruses. Our data provides further evidence of the important role that the WNV / SLEV viral non-structural genetic elements play in viral replication, avian host competence and virulence.

The complexity of circulating HIV type 1 strains in Oyo state, Nigeria.

Multiple HIV-1 subtypes and circulating recombinant forms (CRFs) are known to circulate in West Africa. We undertook a survey of HIVs in Oyo state, in southwestern Nigeria. We analyzed 71 samples from Ibadan, the capital city, and 33 samples from Saki, 100 miles west of Ibadan. We sequenced part of the gag gene and the envelope C2V3 region from 102 and 89 samples, respectively. In the 87 samples for which both genes were sequenced, subtype G and CRF02_AG were found in equal proportions (32.2% each). Other samples included CRF06_cpx (8.0%), subtype A (2.3%), C (1.1%), unclassified (1.1%), or discordant sequences suggesting the presence of a large number of recombinants involving CRF02_AG and/or subtype G (20.7%) or other subtypes (2.3%). The subtype/CRF designation was concordant in two gene fragments in the majority of samples evaluated. However, we observed differences in subtype distribution between the two locations with a predominance of subtype G in Ibadan and CRF02 in Saki. This is the first in-depth analysis of HIV variability at a state level in Nigeria. Our analysis revealed a significant level of viral heterogeneity and a geographical difference in subtype distribution, and demonstrated that CRF02_AG does not account for the majority of circulating strains.

DNA vaccination of the American crow (Corvus brachyrhynchos) provides partial protection against lethal challenge with West Nile virus.

The New York 1999 strain of West Nile virus (WNV) is nearly 100% fatal in the American crow (Corvus brachyrhynchos). We evaluated four WNV vaccine formulations in American crows, including intramuscular (i.m.) DNA vaccine, i.m. DNA vaccine with adjuvant, orally administered microencapsulated DNA vaccine, and i.m. killed vaccine. Neutralizing antibodies developed in approximately 80% of crows that received the DNA vaccine i.m. (with or without adjuvant), and in 44% that received the killed vaccine. However, no crows that received the oral microencapsulated DNA vaccine or the placebo developed WNV antibodies. All crows were challenged 10 wk after initial vaccination. No unvaccinated crows survived challenge, and survival rates were 44% (i.m. DNA vaccine), 60% (i.m. DNA vaccine with adjuvant), 0% (oral microencapsulated DNA vaccine), and 11% (killed vaccine). Peak viremia titers in the birds that survived were significantly lower as compared to titers in birds that died. Parenteral administration of a WNV DNA vaccine was associated with reduced mortality but did not provide sterile immunity.

Antibiotic treatment disrupts bacterial communities in the colon and rectum of simian immunodeficiency virus-infected macaques.

Antibiotic therapies are known to disrupt gastrointestinal (GI) bacterial communities. HIV and pathogenic simian immunodeficiency virus (SIV) infections have also been associated with disrupted GI bacterial communities. We administered a combination antibiotic therapy to six SIV-infected rhesus macaques and collected colon biopsies, stool samples and rectal swabs before and after antibiotics, and evaluated the bacterial communities at each sample site using high-throughput 16S rRNA gene sequencing. The colon mucosa and stool samples displayed different bacterial communities, while the rectal swabs showed a mixture of the mucosal and stool-associated bacteria. Antibiotics disrupted the native bacterial communities at each sample site. The colon mucosa showed depleted abundances of the dominant Helicobacteraceae, while we found depleted abundances of the dominant Ruminococcaceae sp. in the stool. The rectal swabs showed similar trends as the colon mucosa, but were more variable. After the antibiotic treatment, there were increased abundances of similar taxa of facultative anaerobic bacteria, including Lactobacillaceae and Enterobacteriaceae at each sample site.

Subtype-specific patterns in HIV Type 1 reverse transcriptase and protease in Oyo State, Nigeria: implications for drug resistance and host response.

As the use of antiretroviral therapy becomes more widespread across Africa, it is imperative to characterize baseline molecular variability and subtype-specific peculiarities of drug targets in non-subtype B HIV-1 infection. We sequenced and analyzed 35 reverse transcriptase (RT) and 43 protease (PR) sequences from 50 therapy-naive HIV-1-infected Nigerians. Phylogenetic analyses of RT revealed that the predominant viruses were CRF02_AG (57%), subtype G (26%), and CRF06_cpx (11%). Six of 35 (17%) individuals harbored primary mutations for RT inhibitors, including M41L, V118I, Y188H, P236L, and Y318F, and curiously three of the six were infected with CRF06_cpx. Therefore, CRF06_cpx drug-naive individuals had significantly more drug resistance mutations than the other subtypes (p = 0.011). By combining data on quasisynonymous codon bias with the influence of the differential genetic cost of mutations, we were able to predict some mutations, which are likely to predominate by subtype, under drug pressure. Some subtype-specific polymorphisms occurred within epitopes for HLA B7 and B35 in the RT, and HLA A2 and A*6802 in PR, at positions implicated in immune evasion. Balanced polymorphism was also observed at predicted serine-threonine phosphorylation sites in the RT of subtype G viruses. The subtype-specific codon usage and polymorphisms observed suggest the involvement of differential pathways for drug resistance and host-driven viral evolution in HIV-1 CRF02_AG, subtype G, and CRF06_cpx, compared to subtype B. Subtype-specific responses to HIV therapy may have significant consequences for efforts to provide effective therapy to the populations infected with these HIV-1 subtypes.