American Strains Research Articles

Genotype-specific features reduce the susceptibility of South American yellow fever virus strains to vaccine-induced antibodies

The resurgence of yellow fever in South America has prompted vaccination against the etiologic agent, yellow fever virus (YFV). Current vaccines are based on a live-attenuated YF-17D virus derived from a virulent African isolate. The capacity of these vaccines to induce neutralizing antibodies against the vaccine strain is used as a surrogate for protection. However, the sensitivity of genetically distinct South American strains to vaccine-induced antibodies is unknown. We show that antiviral potency of the polyclonal antibody response in vaccinees is attenuated against an emergent Brazilian strain. This reduction was attributable to amino acidchanges at two sites in central domain II of the glycoprotein E, including multiple changes at the domain I-domain II hinge, which are unique to and shared among most South American YFV strains. Our findings call for a reevaluation of current approaches to YFV immunological surveillance in South America and suggest approaches for updating vaccines.

Variability in an effector gene promoter of a necrotrophic fungal pathogen dictates epistasis and effector-triggered susceptibility in wheat.

The fungus Parastagonospora nodorum uses proteinaceous necrotrophic effectors (NEs) to induce tissue necrosis on wheat leaves during infection, leading to the symptoms of septoria nodorum blotch (SNB). The NEs Tox1 and Tox3 induce necrosis on wheat possessing the dominant susceptibility genes Snn1 and Snn3B1/Snn3D1, respectively. We previously observed that Tox1 is epistatic to the expression of Tox3 and a quantitative trait locus (QTL) on chromosome 2A that contributes to SNB resistance/susceptibility. The expression of Tox1 is significantly higher in the Australian strain SN15 compared to the American strain SN4. Inspection of the Tox1 promoter region revealed a 401 bp promoter genetic element in SN4 positioned 267 bp upstream of the start codon that is absent in SN15, called PE401. Analysis of the world-wide P. nodorum population revealed that a high proportion of Northern Hemisphere isolates possess PE401 whereas the opposite was observed in representative P. nodorum isolates from Australia and South Africa. The presence of PE401 removed the epistatic effect of Tox1 on the contribution of the SNB 2A QTL but not Tox3. PE401 was introduced into the Tox1 promoter regulatory region in SN15 to test for direct regulatory roles. Tox1 expression was markedly reduced in the presence of PE401. This suggests a repressor molecule(s) binds PE401 and inhibits Tox1 transcription. Infection assays also demonstrated that P. nodorum which lacks PE401 is more pathogenic on Snn1 wheat varieties than P. nodorum carrying PE401. An infection competition assay between P. nodorum isogenic strains with and without PE401 indicated that the higher Tox1-expressing strain rescued the reduced virulence of the lower Tox1-expressing strain on Snn1 wheat. Our study demonstrated that Tox1 exhibits both ‘selfish’ and ‘altruistic’ characteristics. This offers an insight into a complex NE-NE interaction that is occurring within the P. nodorum population. The importance of PE401 in breeding for SNB resistance in wheat is discussed.

Distribution and genetic diversity of Feline calicivirus in Moscow metropolitan area.

Feline calicivirus (FCV) is widespread throughout the world. An FCV infection is associated with conjunctivitis, rhinitis, and mouth ulcers that can lead to the animal's death. Because vaccination is not always effective, it is necessary to monitor the infection regularly. This study examined the FCV epizootic situation in the Moscow metropolitan area by conducting a molecular phylogenetic analysis of the virus isolates. Samples from 6213 animals were examined by a reverse transcription polymerase chain reaction. For phylogenetic analysis, 12 nucleotide sequences obtained from animal samples were selected. Sequencing was performed using the Sanger method. Phylogenetic analysis was conducted using the Maximum Likelihood method. The FCV genome was detected in 1,596 (25.7%) samples out of 6,213. In 2018, calicivirus was detected in 18.9% of samples, 27.8% in 2019, 21.4% in 2020, and 32.6% in 2021. Phylogenetic analysis of the F ORF2 region and the ORF3 start region led to division into two FCV genogroups. Most of the isolates (8 out of 12) were close to the Chinese strains. On the other hand, there were isolates closely related to European and American strains. The isolates circulating in Moscow were not included in clusters with vaccine strains; their nucleotide similarity varied from 77% to 83%. This study revealed a high prevalence and genetic diversity of the FCV in Moscow. The epizootic situation remains stably tense because 24 viruses were detected in 25% of animals annually.

An overview of Zika virus genotypes and their infectivity.

ABSTRACTZika virus (ZIKV) is an enveloped, single-stranded RNA arbovirus belonging to the genus Flavivirus. It was first isolated from a sentinel monkey in Uganda in 1947. More recently, ZIKV has undergone rapid geographic expansion and has been responsible for outbreaks in Southeast Asia, the Pacific Islands, and America. In this review, we have highlighted the influence of viral genetic variants on ZIKV pathogenesis. Two major ZIKV genotypes (African and Asian) have been identified. The Asian genotype is subdivided into Southwest Asia, Pacific Island, and American strains, and is responsible for most outbreaks. Non-synonymous mutations in ZIKV proteins C, prM, E, NS1, NS2A, NS2B, NS3, and NS4B were found to have a higher prevalence and association with virulent strains of the Asian genotype. Consequently, the Asian genotype appears to have acquired higher cellular permissiveness, tissue persistence, and viral tropism in human neural cells. Therefore, mutations in specific coding regions of the Asian genotype may enhance ZIKV infectivity. Considering that mutations in the genomes of emerging viruses may lead to new virulent variants in humans, there is a potential for the re-emergence of new ZIKV cases in the future.

Enterocytozoon hepatopenaei real-time and Shrimp MultiPathTM PCR assay validation for South-East Asian and Latin American strains of Penaeid shrimp.

Enterocytozoon hepatopenaei (EHP) infections are a global challenge for the Penaeid shrimp industry with a sharp rise in prevalence over the last 10 yr. EHP is known to cause sub-optimal growth, large size variation and reduced survival of shrimp. Molecular methods development has mainly focussed on 18S rRNA or spore wall protein 1 (SWP1). Due to the specificity and sensitivity issues with previously designed assays for both targets, new molecular assays are needed by the global shrimp industry and regulators to help manage the risks posed by EHP. This paper describes new real-time PCR (qPCR) methods developed for the novel EHP gene targets polar tube protein 2 (PTP2) and spore wall protein 26 (SWP26), whilst also presenting performance metrics of the new Shrimp MultiPathTM technology EHP assay. qPCR assays PTP2G and SWP26G show high amplification efficiency, a limit of detection (LOD) of between 1 and 4 copies, low assay variation and high diagnostic sensitivity (DSe) and specificity (DSp) compared to imperfect reference assays. Similar performance is seen with Shrimp MultiPathTM EHP showing an LOD of 8 copies, low assay variation and high DSe and DSp. These novel molecular targets for EHP and Shrimp MultiPathTM EHP strengthen global efforts to monitor and mitigate risks of EHP infections and outbreaks. Moreover, this study presents novel data on distribution of EHP in shrimp populations from South-East Asia and Latin America, and how sequence variations need to be considered when monitoring EHP in different geographies.

Molecular Phylogenetic Study of Strains Morphologically Identified as <i>Exophiala dermatitidis</i> from Clinical and Environmental Specimens in Japan

In this study, we aimed to clarify the phylogenetic distribution of Exophiala dermatitidis in Japan and describe the characteristics of genotypes. We examined 67 clinical and environmental isolates that were morphologically identified and preserved as E. dermatitidis and we confirmed the identification on the basis of the ribosomal DNA internal transcribed spacer region. Genotype sequences were aligned and compared using maximum likelihood phylogenetic tree analyses of the ITS1 region. Additionally, the strains of each genotype were tested for mycological characteristics, such as growth temperature, growth rate, and drug sensitivity. The 67 strains examined were isolated from Japan, the United States, Brazil, Venezuela, and China. In accordance with the establishment of a phylogenetic tree for the ITS1 region, 45 of the 49 Japanese strains were classified as genotype A, two as genotype B, and two as genotype D (A2 according to the method of Matos et al. (2003)). Chinese strains were divided into genotypes A and D (A2), and South American strains were classified into genotypes A, B, B2, and C2, while all strains from the U.S. belonged to genotype A. New genotype groups B2 and C2 were identified in Brazilian and Venezuelan strains, respectively. There were no specific differences among the genotypes or isolated regions in the antifungal susceptibility test for all E. dermatitidis isolates. However, genotypes B2 and D (A2) exhibited growth at higher temperatures than the other genotypes.

Genetic and Antigenic Diversity of Infectious Bronchitis Virus in South America.

Infectious bronchitis virus (IBV) is one of the economically most important diseases affecting the South American poultry industry. The extensive genomic heterogeneity of IBV is a consequence of high mutation rates and recombination events followed by selection. Nucleotide heterogeneity is much higher in the S1 coding region of the relevant spike protein; thus, the S1 sequence is widely used for the IBV genetic classification in genotypes and lineages. Two main lineages (GI-11 and GI-16) extensively circulate in South American chicken flocks. The GI-11 lineage, found exclusively in South America, emerged in the 1950s and is currently the predominant lineage in Brazil and Uruguay. The GI-16 lineage emerged around 1979 and is now circulating in most South American regions. All South American countries include Massachusetts-type strains (GI-1 lineage) in the IBV vaccination programs. The GI-11 and GI-16 lineages display very low antigenic relatedness to Massachusetts vaccine strains. Because these vaccine strains may not confer complete protection against South American lineages, other vaccination strategies have been reported to control GI-11 and GI-16 outbreaks. Analysis of the few full-length genomes of South American strains highlights a complex recombination history of IBV in the continent. A broader geographic and temporal sampling is needed to understand the pattern of genetic variability and the evolutionary history of IBV variants in South America.

Isolation and Molecular Characteristics of a Novel Recombinant Avian Orthoreovirus From Chickens in China.

In recent years, the emergence of avian orthoreovirus (ARV) has caused significant losses to the poultry industry worldwide. In this study, a novel ARV isolate, designated as AHZJ19, was isolated and identified from domestic chicken with viral arthritis syndrome in China. AHZJ19 can cause typical syncytial cytopathic effect in the chicken hepatocellular carcinoma cell line, LMH. High-throughput sequencing using Illumina technology revealed that the genome size of AHZJ19 is about 23,230 bp, which codes 12 major proteins. Phylogenetic tree analysis found that AHZJ19 was possibly originated from a recombination among Hungarian strains, North American strains, and Chinese strains based on the sequences of the 12 proteins. Notably, the σC protein of AHZJ19 shared only about 50% homology with that of the vaccine strains S1133 and 1733, which also significantly differed from other reported Chinese ARV strains. The isolation and molecular characteristics of AHZJ19 provided novel insights into the molecular epidemiology of ARV and laid the foundation for developing efficient strategies for control of ARV in China.

Isolation and Phylogenetic Analysis of Reemerging Pseudorabies Virus Within Pig Populations in Central China During 2012 to 2019.

To understand the biological characteristics of the reemerging pseudorabies virus (PRV) strains, a total of 392 tissue samples were collected from diseased pigs during reemerging PR outbreaks between 2012 and 2019 on farms in central China where swine had been immunized with Bartha-K61 and 51 (13. 01%) were positive for the gE gene by PCR. Sixteen PRV strains were isolated and caused clinical symptoms and death in mice. Subsequently, gE, gC, gB, and gD complete genes were amplified from the 16 PRV isolates and sequenced. Phylogenetic analysis based on these four gene sequences shows that the 16 PRV isolates were more closely related to the Chinese PRV variants (after 2012) but genetically differed from early Chinese PRV isolates (before 2012). Sequence analysis reveals that PRV isolates exhibited amino acid insertions, substitutions, or deletions compared with early Chinese PRV isolates and European–American PRV strains. In addition, this is the first report that eight isolates (8/16) in this study harbor a unique amino acid substitution at position 280 (F to L) of the gC protein, and six isolates have an amino acid substitution at position 338 (A to V) of the gD protein compared with the Chinese PRV variants. The emulsion containing inactivated PRV NY isolate could provide complete protection against the NY isolate. This study might enrich our understanding of the evolution of reemerging PRV strains as well as pave the way for finding a model virus to develop a novel vaccine based on reemerging PRV strains.

Changes in the spike and nucleocapsid protein of porcine epidemic diarrhea virus strain in Vietnam-a molecular potential for the vaccine development?

BackgroundPorcine epidemic diarrhea virus (PEDV) is a dangerous virus causing large piglet losses. PEDV spread rapidly between pig farms and caused the death of up to 90% of infected piglets. Current vaccines are only partially effective in providing immunity to suckling due to the rapid dissemination and ongoing evolution of PEDV.MethodsIn this study, the complete genome of a PEDV strain in Vietnam 2018 (IBT/VN/2018 strain) has been sequenced. The nucleotide sequence of each fragment was assembled to build a continuous complete sequence using the DNASTAR program. The complete nucleotide sequences and amino acid sequences of S, N, and ORF3 genes were aligned and analyzed to detect the mutations.ResultsThe full-length genome was determined with 28,031 nucleotides in length which consisted of the 5′UTR, ORF1ab, S protein, ORF3, E protein, M protein, N protein, and 3′UTR region. The phylogenetic analysis showed that the IBT/VN/2018 strain was highly virulent belonged to the G2b subgroup along with the Northern American and Asian S-INDEL strains. Multiple sequence alignment of deduced amino acids revealed numerous mutations in the S, N, and ORF3 regions including one substitution 766P > L766 in the epitope SS6; two in the S0subdomain (135DN136 > 135SI136 and N144> D144); two in subdomain SHR1 at aa 1009L > M1009 and 1089S > L1089; one at aa 1279P > S1279 in subdomain SHR2 of the S protein; two at aa 364N > I364 and 378N > S378 in the N protein; four at aa 25L > S25, 70I > V70, 107C > F107, and 168D > N168 in the ORF3 protein. We identified two insertions (at aa 59NQGV62 and aa 145N) and one deletion (at aa 168DI169) in S protein. Remarkable, eight amino acid substitutions (294I > M294, 318A > S318, 335V > I335, 361A > T361, 497R > T497, 501SH502 > 501IY502, 506I > T506, 682V > I682, and 777P > L777) were found in SA subdomain. Besides, N- and O-glycosylation analysis of S, N, and ORF3 protein reveals three known sites (25G+, 123N+, and 62V+) and three novel sites (144D+, 1009M+, and 1279L+) in the IBT/VN/2018 strain compared with the vaccine strains. Taken together, the results showed that mutations in the S, N, and ORF3 genes can affect receptor specificity, viral pathogenicity, and the ability to evade the host immune system of the IBT/VN/2018 strain. Our results highlight the importance of molecular characterization of field strains of PEDV for the development of an effective vaccine to control PEDV infections in Vietnam.

Phylogenetic Differentiation of Rickettsia parkeri Reveals Broad Dispersal and Distinct Clustering within North American Strains

ABSTRACTThe tick-borne pathogen Rickettsia parkeri causes a mild rickettsiosis, with cases reported from several countries to its known distribution in the Americas. Molecular analyses have identified a clear distinction between strains of R. parkeri sensu stricto (s. s.) and R. parkeri sensu lato (s. l.) as well as separation between North American and South American R. parkeri s. s. strains. To expand on this previous work, we developed a multilocus sequence typing analysis with two aims: first, to investigate the genetic diversity within strains of North American R. parkeri s. s., and second, to further the understanding of the genetic relationships between R. parkeri s. s. and R. parkeri s. l. Sixty-four R. parkeri isolates and 12 R. parkeri-positive tick lysates were analyzed using a novel typing scheme consisting of four coding regions and two intergenic regions. A concatenated Bayesian phylogeny that identified eight clades was constructed: three represent the R. parkeri s. l. strains, and five represent the R. parkeri s. s. strains. The clades appear to be generally phylogeographically organized and associated with specific tick vectors. However, while one of the four R. parkeri s. s. North American clades appears to be limited to the southwestern United States, the other North American clades exhibit broad dispersal, most notably seen in the largest group, which includes representative samples extending from northern Mexico to Delaware. This work highlights the increasingly recognized geographic range of R. parkeri in the Americas and suggests a potential public health risk for these areas.IMPORTANCE Since 1937, when Rickettsia parkeri was originally identified in Amblyomma maculatum group ticks, the recognized range and associated vectors for this pathogen have expanded significantly. In recent years, R. parkeri has been identified in 12 tick species from seven countries in the Americas. Herein, we provide evidence that the greatest genetic diversity within R. parkeri exists in North America, where one R. parkeri sensu lato and four R. parkeri sensu stricto genotypes are present. While one distinct R. parkeri sensu stricto genotype exists only in the southwestern United States, three genotypes are broadly distributed in the eastern United States, with the largest of these found across the known range of R. parkeri in North America. In contrast, the South American R. parkeri sensu stricto samples represent a single genotype and are completely clonal at the loci analyzed, irrespective of their country of origin.

Leu-to-Phe substitution at prM146 decreases the growth ability of Zika virus and partially reduces its pathogenicity in mice

Zika virus (ZIKV) is a mosquito-borne flavivirus that causes febrile illness. The recent spread of ZIKV from Asia to the Americas via the Pacific region has revealed unprecedented features of ZIKV, including transplacental congenital infection causing microcephaly. Amino acid changes have been hypothesized to underlie the spread and novel features of American ZIKV strains; however, the relationship between genetic changes and the epidemic remains controversial. A comparison of the characteristics of a Southeast Asian strain (NIID123) and an American strain (PRVABC59) revealed that the latter had a higher replication ability in cultured cells and higher virulence in mice. In this study, we aimed to identify the genetic region of ZIKV responsible for these different characteristics using reverse genetics. A chimeric NIID123 strain in which the E protein was replaced with that of PRVABC59 showed a lower growth ability than the recombinant wild-type strain. Adaptation of the chimeric NIID123 to Vero cells induced a Phe-to-Leu amino acid substitution at position 146 of the prM protein; PRVABC59 also has Leu at this position. Leu at this position was found to be responsible for the viral replication ability and partially, for the pathogenicity in mouse testes.

First Report of Alfalfa Anthracnose Caused by Colletotrichum americae-borealis in Xinjiang, China.

Alfalfa (Medicago sativaL.) is widely planted in the world as one of the most important leguminous forage crops, and it is also the first choice of forage crops for animal feed in Xinjiang. In June 2018, alfalfa plants with typical anthracnose symptoms were observed in 75% of alfalfa fields in Hutubi County, Xinjiang, China. The disease usually occurred in alfalfa fields that had been planted for more than 2 years and was distributed in patches in the field. The incidence rate ranged from 7.5% to 53%, and the fatality rate ranged from 0 to 3%. Greater incidence was observed in fields with older stands. At the early stage of disease, pale brown prismatic or oval sunken lesions with dark brown to black edges were observed at the base of the stem of alfalfa plants. As the symptoms progressed, lesions on stems turned necrotic, and the center of the lesion became gray-white with black dots. In severe cases, the lesion expanded around the stem, causing the upper part of the stem to break off, or wilt and die. Twenty plant stem sections with typical symptoms were sampled and surface-sterilized with 75% ethanol for 30 s and 1% NaClO for 1 min, rinsed in sterilized distilled water, dried on sterilized filter paper for 45 s, placed on potato dextrose agar (PDA), and incubated in the dark at 25°C for 7 days. A fungus was frequently isolated from the surface-sterilized segments, and the colonies of this fungus were white and flat at first, and later the center of colonies became pale brown with black microsclerotia (2.0~3.2 mm. n = 30) and white or brown acervuli (1.0~1.8 mm. n = 30). A large number of conidia and setae spread from ruptured microsclerotia under microscopy. Conidia (n = 40) were hyaline, smooth-walled, straight, aseptate, cylindrical to fusoid, both tips acute to round, 13.7 to 19.5×3.0 to 4.5 μm . Setae (n = 30) were dark brown to black, smooth-walled, 3~6 septate, straight or slightly curved, 66.9~185.1 μm long, tip round and base swollen, 3.9~5.2 μm width. Sometimes setae formed directly on hyphae or brown acervuli. Colony and conidia morphology were similar to the description of Colletotrichum americae-borealis (Damm et al. 2014; Lyu et al. 2020). DNA was extracted from fresh mycelia of three representative isolates (R11, R12 and R13) and the ITS, ACT, CHS-1 and HIS3 genes of three isolates were amplified and sequenced using the primers described previously by Damm et al. (2014). The sequences of three isolates were identical, and twelve aligned sequences from three representative isolates were deposited in GenBank (MT877442, MT877443 and MT877444 for ITS, MW854350, MW854351 and MW854352 for ACT, MW270930, MW270931 and MW270107 for CHS-1, MW854347, MW854348 and MW854349 for HIS3). Sequence analysis revealed that the ITS, ACT, CHS-1 and HIS3 sequences of three representative isolates were shared 99% (355/356 bp for HIS3) to 100% (550/550 bp for ITS, 261/261 bp for ACT, 221/221 bp for CHS-1) identities to each sequence of an American strain (CBS 136232) of C. americae-borealis from alfalfa in GenBank (NR160760 for ITS, KM105434 for ACT, KM105294 for CHS-1, KM105364 for HIS3). Four phylogenetic trees were constructed by the Mrbayes method (Damm et al. 2014), and the result showed that three representative isolates grouped with C. americae-borealis. Combined with morphological observation and molecular biological identification, the pathogen was identified as C. americae-borealis. Pathogenicity tests were executed twice on alfalfa seedlings in a greenhouse. Pots containing ten 40-day old seedlings (Xinjiang daye) were sprayed with a 100 ml of condial suspension (10^6 condia/ml) of R11. Control pots were sprayed with 100 ml of sterile distilled water. Two weeks after inoculation under greenhouse conditions (25 ± 2°C, 12-h photoperiod, 85% humidity), brown spots and necrotic lesions developed on the stem of inoculated alfalfa seedlings, which were similar to disease plants in fields, and C. americae-borealis was reisolated from symptomatic tissue. The control seedlings remained symptomless. Anthracnose caused by C. americae-borealis was reported on alfalfa in the north region of America and Iran (Damm et al. 2014; Alizadeh et al. 2015), as well as Gansu, Inner Mongolia, Yunnan and Heilongjiang Province of China ( Xu. 2019; zhang et al. 2020) . To our knowledge, this is the first report of Colletotrichum americae-borealis causing Alfalfa Anthracnose in Xinjiang, China. This finding can provide an important reference for understanding the distribution and control of this disease.

Polymorphisms in HTLV-1 Tax-responsive elements in HTLV-1-associated myelopathy/tropical spastic paraparesis patients are associated with reduced proviral load but not with disease progression.

Human T-lymphotropic virus type 1 (HTLV-1) provirus expression is mainly directed by Tax-responsive elements (TRE) within the long terminal repeats (LTR). Mutations in TRE can reduce provirus expression and since a high proviral load (PVL) is a risk factor for the development of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), we evaluated polymorphisms in the 5' LTR and the association with PVL and disease progression. HTLV-1 LTR and tax sequences derived from asymptomatic carriers (AC) and HAM/TSP patients followed in a longitudinal study were analysed according to PVL and clinical severity. Individuals infected with HTLV-1 presenting the canonical TRE, considering strain ATK-1 as the consensus, displayed sustained higher PVL. By contrast, an LTR A125G mutation in TRE was associated with slightly reduced PVL only in HAM/TSP patients, although it did not influence the speed of disease progression. Moreover, this polymorphism was frequent in Latin American strains of the HTLV-1 Cosmopolitan Transcontinental subtype. Therefore, polymorphisms in the 5' TRE of HTLV-1 may represent one of the factors influencing PVL in HAM/TSP patients, especially in the Latin American population. Indeed, higher PVL in the peripheral blood has been associated with an increased inflammatory activity in the spinal cord and to a poorer prognosis in HAM/TSP. However, this event was not associated with TRE polymorphisms.

Prevalence, molecular detection, and pathological characterization of small ruminant lentiviruses in goats from Mexico

Small ruminant lentiviruses (SRLVs) are one of the main viral agents affecting goats worldwide. They cause a systemic, chronic, incurable, degenerative disease. In Mexico, the B1 genotype of SRLV has been detected as endemic in goats and sheep; however, its pathological characteristics have not yet been evaluated. In this study, sampling was performed to identify the percentage of SRLV positivity in different farms in Mexico. Prevalence rates of 19.6 % and 11.6 % were detected through PCR and serology, respectively. Through a real-time PCR differential test, the prevalence of genotype B was identified. From a chosen case, an SRLV strain was isolated (which was called SRLV/B1/Goat/Mx/INIFAP-1/2013). The phylogenetic analysis revealed that the strain SRLV/B1/Goat/Mx/INIFAP-1/2013 shares a great homology with an American and Asiatic strains of the B1 genotype. Virulence of the isolated strain was analyzed during an experimental infection in kids. The kids did not present clinical disease after the experimental infection. The high viral load values persisted throughout the entire experiment (90 days), and no seroconversion was detected. At necropsy, macroscopic alterations were not observed, except in the lungs. The microscopic lesions in the respiratory tract consisted of moderate interstitial pneumonia, peribronchial lymphoid hyperplasia, and bronchial exudate. In the central nervous system, moderate gliosis, meningeal congestion, lymphocyte infiltration, satellitosis, and demyelination were observed. The virus was systemically distributed among organs; the cerebellum and carpal synovial fluid samples were those with the highest viral load, 5.73 copies of virus per ng of total DNA (log10) and 5.07 log10, respectively. In this study, we identified a strain with high homology to Asiatic strains that manifested low virulence in kids during short infection. Understanding the strains that circulate in the goat population will allow implementing better diagnostic methodologies to propose disease control alternatives for this emerging animal production system in Mexico.

Family-centered prevention buffers the effect of financial strain on parenting interactions, reducing youth conduct problems in African American families.

This study examined whether a family-focused prevention program for African American families could buffer the negative effect of perceived financial strain on protective parent-child interactions and thereby reduce the indirect effect of financial strain on youth conduct problems. Three hundred and forty-six African American couples with an early adolescent child (M age = 10.9 years at Wave 1 [W1]) participated. Families were randomly assigned to the Protecting Strong African American Families (ProSAAF) prevention program or to a no-treatment control condition and provided data prerandomization (W1) and postintervention, Wave 2 (W2), 9.4 months after W1. Youth and both parents reported family financial strain at W1 and protective parent-child interactions at W1 and W2. Youth reported their conduct problems at W1 and W2. Hypotheses were tested using moderated mediation analyses. ProSAAF significantly moderated the negative effect of youth-reported financial strain on changes in protective parenting interactions such that financial strain was not significantly associated with changes in protective parenting among the intervention group, thus reducing the indirect effect of financial strain on increases in youth conduct problems among this group. Parent-reported financial strain was not significantly associated with changes in protective parenting in either condition, precluding the potential for stress buffering intervention effects. The ProSAAF prevention program buffered the negative effect of youth-reported financial strain on protective parenting interactions and reduced its indirect effect on youth conduct problems. These findings suggest that family-focused prevention has the potential to promote resilience among African American youth perceiving family financial strain. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

A next generation sequencing (NGS) analysis to reveal genomic and proteomic mutation landscapes of SARS-CoV-2 in South Asia.

Counts for SARS-CoV-2 associated infections and fatalities are on the rise globally even in regions which contained the spread momentarily. The pattern of infections has been found to be controlled by the distinctive selection pressures exerted by fluctuating environmental nature and hosts. A total of 410 whole-genome sequences submitted by the South Asian countries were retrieved from the GISAID database and analyzed to assess the impact and pattern of mutations in this region. Most common and frequent mutations in the South Asian countries are 241C>T, 3037C>T, 14408C>T, and 23403A>G and about 85% SNPs are localized in ORF1ab, spike protein, and nucleocapsid. Among the identified mutations, the proportion of missense type (54.17%) was highest, followed by the synonymous (41.66%) and the non-coding types (4.17%). While analyzing transmission source in terms of geolocation, the largest clustered group from the South Asian countries was based on the G-clade (D614G) (81.7%; 335/410 samples), tracing the inception and transmission of SARS-CoV-2 infections in the South Asian countries from European regions. Phylogenetic analysis also revealed that the South Asian strains are highly related to the South American and European strains. We found that G-clade mutations are more prevalent (96.19%) in the samples of Bangladesh which were also prevalent in the European isolates. Surprisingly, one missense mutation (1163A>T) in ORF1ab gene became dominant only in Bangladesh (78.8%), which led to debates regarding effects on the pathogenicity and transmissibility of the virus. Overall, the findings of this study highlight the frequently mutated SARS-CoV-2 variants among the COVID-19 patients in the South Asian countries which might ease containment of the disease in this region through investigating the virulence reducing factors as the identified mutations are strongly correlated with low infection and mortality rate.

Death From Primary Amebic Meningoencephalitis After Recreational Water Exposure During Recent Travel to India-Santa Clara County, California, 2020.

In February 2020, a man returned to the United States after an 11-day trip to India and died of primary amebic meningoencephalitis (PAM), caused by nasal exposure to the free-living ameba Naegleria fowleri found in warm water. We identified potential exposures, confirmed etiology, and described the molecular epidemiology of the infection. We reviewed medical records to describe his clinical course and interviewed his family to determine water exposures. Genotyping was performed on the N. fowleri strain and compared with North American strains through repetitive nonpolymorphic nuclear loci analysis to identify differences. We reviewed N. fowleri strains in the National Center for Biotechnology Information database (GenBank) to determine genotypes present in India. The patient became acutely encephalopathic 3 days after returning; the only known nasal water exposure was at an indoor swimming pool in India 5 days earlier. Cerebrospinal fluid (CSF) testing demonstrated neutrophil-predominant pleocytosis and low glucose, but negative gram stain and culture. CSF microscopy revealed trophozoites; N. fowleri was detected by real-time polymerase chain reaction. Classical genotyping confirmed genotype I, common in the United States and among Indian strains in GenBank. The North American N. fowleri strains and the patient's strain varied at 5 nonpolymorphic loci. A man died from PAM after likely exposure at a vacation rental pool in India. We recommend including PAM in the differential diagnosis when CSF studies suggest bacterial meningitis but gram stain is negative. Genotyping can advance our understanding of N. fowleri molecular epidemiology and support future investigations.

Two chemoattenuated PfSPZ malaria vaccines induce sterile hepatic immunity.

The global decline in malaria has stalled1, emphasizing the need for vaccines that induce durable sterilizing immunity. Here we optimized regimens for chemoprophylaxis vaccination (CVac), for which aseptic, purified, cryopreserved, infectious Plasmodium falciparum sporozoites (PfSPZ) were inoculated under prophylactic cover with pyrimethamine (PYR) (Sanaria PfSPZ-CVac(PYR)) or chloroquine (CQ) (PfSPZ-CVac(CQ))-which kill liver-stage and blood-stage parasites, respectively-and we assessed vaccine efficacy against homologous (that is, the same strain as the vaccine) and heterologous (a different strain) controlled human malaria infection (CHMI) threemonths after immunization( https://clinicaltrials.gov/ , NCT02511054 andNCT03083847). We report that a fourfold increase in the dose of PfSPZ-CVac(PYR) from 5.12×104 to 2×105 PfSPZs transformed a minimal vaccine efficacy (low dose, two out of nine (22.2%) participants protected against homologous CHMI), to a high-level vaccine efficacy with seven out of eight (87.5%) individuals protected against homologous and seven out of nine (77.8%) protected against heterologous CHMI. Increased protection was associated with Vδ2 γδ T cell and antibody responses. At the higher dose, PfSPZ-CVac(CQ) protected six out of six (100%) participants against heterologous CHMIthreemonths after immunization. All homologous (four out of four) and heterologous (eight out of eight) infectivity control participants showed parasitaemia. PfSPZ-CVac(CQ) and PfSPZ-CVac(PYR) induced a durable, sterile vaccine efficacy against a heterologous South American strain of P. falciparum, which has a genome and predicted CD8 T cell immunome that differs more strongly from the African vaccine strain than other analysed African P. falciparum strains.

Zika Virus Infection Activates Proinflammatory Cytokines and Triggers Monocyte Differentiation

In 2015, Zika virus (ZIKV) outbreak in several countries of South America was linked to microcephaly in newborns. Microcephaly, a severe clinical presentation, was first diagnosed in association with ZIKV, which primarily considered self-limiting infection. From infected keratinocytes, virus disseminates crossing blood-tissue barriers and is detected in various tissues including brain, muscles and placenta. One of the mechanisms utilized by viruses to cross tissue barriers involves leukocytes, where monocytes are often identified as the carriers. Recently, circulating monocytes were shown to harbor ZIKV, suggesting that these leukocyte populations could serve as vehicle to transport ZIKV to the fetus. We speculate that ZIKV infection could promote monocyte activation and release of cytokines causing tissue damage. However, little is known about monocyte susceptibility to ZIKV infection. Also, effect of ZIKV infection on transcriptional activation and production remains unclear.Materials and methods. Monocytes were isolated from cord blood buffy coats (University of Colorado Cord Blood Bank, Aurora) using Miltenyi magnetic bead separation kit (CD14 Microbeads; Miltenyi, Auburn, CA). Monocytes were infected with two ZIKV strains, PRVABC59 (South America) and IBH30656 (Nigeria), ATCC (Manassas, VA). Total RNA was extracted and used for Next generation Sequencing (Illustra RNAspin Mini kit (GE Healthcare, Marlborough, MA)). FastQ data generated by the MiSeq (Illumina, San Diego CA) was annotated and the sequence reads were analyzed by CLC workbench 9.0.1 (Qiagen, Germantown, MD) for the detection of viral and cellular genes. Differential expressions of cellular genes based on the Reads Per Kilobase of transcript per Million mapped reads were determined by comparing with mock-infected cells using RNA-seq analysis tool of CLC Workbench. Differentially regulated genes due to ZIKV infection, identified by CLC Workbench, were subjected for determining the deregulated pathways using IPA Ingenuity Pathways analysis tool (Qiagen Germantown, MD). Viral replication was determined by qPCR, Western blot and immunofluorescence. Cytokine activation was analyzed using Bio-Plex Pro Human Cytokine 27-plex Panel (Bio-Rad, Hercules, CA, USA) following manufacturer's instructions.Results. We have shown that South American and Nigerian strains of ZIKV can infect monocytes and actively replicate. Presence of capsid protein and accumulation of viral transcripts was demonstrated in vitro. Infection with PRVABC59 strain changes transcriptional activity of 332 cellular genes, while the Nigerian strain affected the transcription of 521 cellular genes compared to the mock-infected controls. Interestingly, 195 genes were commonly deregulated in cells infected with both strains. Analysis of these commonly affected genes identified multiple pathways including apoptosis, chemotaxis, cell proliferation, inflammation, acute phase response, mitochondria activation. The most amazing observation was upregulation of mitochondrial and inflammasome genes. Mitochondrial genes (MT-ND2, MT-ND5 and MT-ATP8) activated in ZIKV infected monocytes were shown to play a key role in pathogenesis of other neurodegenerative disease.Analysis of cytokine production revealed early activation of proinflammatory cytokines (IL-1β, IL-6 and IL-8) (Table 1). Upregulation of IL-1β by ZIKV infected monocytes is evident for inflammasome activation. Upregulation of IL-6, IL-10 and IL-17α suggested an activation of Th17 pathway in ZIKV infected monocytes. Besides only Nigerian ZIKV strain increased the production of IL-4 in monocytes. IL-4 together with IL-10 could promote Th2 type immune response to ZIKV. Finally, both strains increased production of FGF, while levels of VEGF were increased only in supernatants of PRVABC59 infected monocytes.Conclusions. Human monocytes are susceptible to infection with ZIKV strains, PRVABC59 and IBH30656 and support viral replication. ZIKV infection affects cellular pathways, including mitochondrial and inflammasome ones that could play significant role in pathogenesis. ZIKV infection of monocytes upregulates cytokine production, which can trigger inflammation and leukocyte migration. Analysis of cytokine production by ZIKV infected monocytes suggests activation of Th2 and Th17 leukocytes, supporting the role of this signaling in ZIKV pathogenesis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.