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Koala retrovirus (KoRV), a major pathogen of koalas, exists in both endogenous (KoRV-A) and exogenous forms (KoRV-B to J). However, the impact of infection with multiple subtypes is not well understood. Accordingly, in this study, we surveyed a representative sample from a Japanese zoo population to determine the infection status for three KoRV subtypes (KoRV-A, B, and C) and to investigate the proviral and RNA load profiles in animals with single- and multiple-subtype infections, using peripheral blood mononuclear cells (PBMCs) and plasma. Six koalas were evaluated in the study; all were infected with KoRV-A, and two koalas were coinfected with non-A subtypes (KoRV-B and/or KoRV-C). The highest KoRV total RNA and viral loads in PBMCs and plasma were found in a koala infected with multiple subtypes (KoRV-A, -B and -C). The other koala infected with multiple subtypes (KoRV-A and B) showed the highest proviral PBMC load but the lowest RNA copy number in PBMC and plasma. PBMCs from this animal were cultured for further investigation, and KoRV RNA was detected in the cells and culture supernatant after 7 and/or 14 days. The koalas harboring multiple subtypes had a higher white blood cell count than those harboring only KoRV-A and were judged to be leukemic, and they subsequently died due to lymphoma. Accordingly, we conclude that coinfection with multiple KoRV subtypes may be linked to more-severe disease. In a sequence alignment, the detected KoRV-A env gene showed 100% sequence identity to the reference gene, whereas the KoRV-B and -C env genes varied from their reference sequences.

In vitro cell cultures are widely used models for dissecting cellular and molecular mechanisms that lead to certain physiological conditions and diseases. The pathogenesis of BoHV-4 in the bovine reproductive tract has been studied by conducting tests on primary cultures. However, many questions remain to be answered about the role of BoHV-4 in endometrial cells. The aim of this study was to compare the replication and gene expression of BoHV-4 in cell lines and bovine reproductive tract primary cells as an in vitro model for the study of this virus. We demonstrated that BoHV-4 strains differ in their in vitro growth kinetics and gene expression but have the same cell type preference. Our results demonstrate that BoHV-4 replicates preferentially in bovine endometrial cells (BEC). However, its replication capacity extends to various cell types, since all cells that were tested were permissive to BoHV-4 infection. The highest virus titers were obtained in BEC cells. Nevertheless, virus replication efficiency could not be fully predicted from the mRNA expression profiles. This implies that there are multiple cell-type-dependent factors and strain properties that determine the level of BoHV-4 replication. The results of this study provide relevant information about the in vitro behavior of two field isolates of BoHV-4 in different cell cultures. These findings may be useful for the design of future in vitro experiments to obtain reliable results not only about the pathogenic role of BoHV-4 in the bovine female reproductive tract but also in the development of efficient antiviral strategies.

South Texas has experienced local transmission of Zika virus and of other mosquito-borne viruses such as chikungunya virus and dengue virus in the last decades. Using a mosquito surveillance program in the Lower Rio Grande Valley (LRGV) and San Antonio, TX, from 2016 to 2018, we detected the presence of an insect-specific virus, cell fusing agent virus (CFAV), in the Aedes aegypti mosquito population. We tested 6,326 females and 1,249 males from the LRGV and 659 females from San Antonio for CFAV by RT-PCR using specific primers. Infection rates varied from 0 to 261 per 1,000 mosquitoes in the LRGV and 115 to 208 per 1,000 in San Antonio depending on the month of collection. Infection rates per 1,000 individuals appeared higher in females collected from BG Sentinel 2 traps compared to Autocidal Gravid Ovitraps, but the ratio of the percentage of infected pools did not differ by trap type. The natural viral load in individual males ranged from 1.25 x 10 2 to 5.50 x 10 6 RNA copies and in unfed females from 5.42 x 10 3 to 8.70 x 10 6 RNA copies. Gravid females were found to harbor fewer viral particles than males and unfed females.

In South Africa (SA), hepatitis B virus (HBV) infection is strongly associated with hepatocellular carcinoma (HCC). As HBV genotypes/subgenotypes and mutations can influence disease manifestation and progression, our aim was to molecularly characterize HBV in Black cancer patients, with and without HCC. The basal core promoter/precore (BCP/PC) and complete surface (S) regions of HBV isolates were amplified and sequenced from 55 HCC cases and 22 non-HCC cancer controls. Phylogenetic analysis of 43 polymerase/complete S region amplicons showed that the majority (88.4%) clustered with subgenotype A1, 4.7% with A2, and 7% with A3. The following mutations were significantly more frequent in HCC cases than in controls (p < 0.05): in the BCP/PC 1753C/G (22.5% vs. 0%), 1764A (69.4% vs. 38.1%), and T64C (51.5% vs. 20%) in the preS2, which results in a F22L substitution. PreS1 and preS2 start codon mutants were detected only in HCC cases, occurring in two and 16 isolates, respectively. PreS deletion mutants were isolated from 11 HCC cases, which had a HBV viral load > 10,000 IU/mL and were significantly younger than non-HCC controls (34 ± 7.1 vs. 41.2 ± 9.5 years, p = 0.05). The 1762T/1764A double mutation was detected in the majority (90.9%) of the isolates from HCC cases with preS deletions. Black HBV carriers were mainly infected with subgenotype A1, with HCC cases carrying BCP/PC and preS mutant strains that are associated with hepatocarcinogenesis. This is the first study to compare the molecular characteristics of HBV from HCC and non-HCC cancer patients in SA.

In this study, we compared the characteristics of two strains of Zika virus (ZIKV) isolated in Thailand, one isolated from a febrile patient and one isolated from tissues of a fetus medically terminated due to congenital Zika syndrome (CZS). Replication profiles showed that the isolate from the fetal tissues replicated significantly more slowly than the fever-associated isolate in human lung A549 cells during the first 24 hours postinfection but showed a similar growth profile over longer-term infection. A much smaller difference was observed in Aedes albopictus C6/36 cells. In a quasispecies analysis, a high proportion (approximately 20%) of nonfunctional genomes was identified, caused by an adenine insertion in the prM gene. This insertion was found to be present in two Thai fever strains and as such may represent a common feature of Thai endemic ZIKV. Comparison between viral RNA copy number and viral titer showed that the isolate from fetal tissues was produced more efficiently than the fever-associated isolate. Together, these results suggest that different ZIKV isolates differ in their replication capacity, and this might contribute to the fetotropic potential of a particular strain.

We found a HLA class II histocompatibility antigen gene, DQ alpha 1 chain (HLA-DQA1), that was expressed more than 9-fold higher in high-load hepatitis C virus (HCV) livers than low-load HCV livers using transcriptomics of chronic HCV-infected livers. To further investigate this finding, we examined which cells were positive for HLA-DQA1 and what liver immune responses were different between HCV-high and -low livers. HLA-DQA1-positive cells were significantly increased in the HCV-high group, and most positive cells were identified as non-parenchymal sinusoid cells and lymphocytic infiltrates in the portal area. Parenchymal hepatocytes were negative for HLA-DQA1. HLA-DQA1-positive cells in the liver sinusoid were positive for CD68 (macrophages or Kupffer cells); those in the lymphocytic infiltrates were positive for CD20 (B cells) or CD3 (T cells). mRNA levels of antigen-presenting cell (APC) markers such as CD68 and CD11c were significantly upregulated in the HCV-high group and were correlated with HLA-DQA mRNA levels. CD8B mRNA (CD8 + T cells) was upregulated in both HCV-positive livers compared with HCV-negative livers, whereas CD154 mRNA (CD4 + T helper cell) was upregulated in the HCV-high group compared with the HCV-low group. The immune regulatory molecules FOXP3 mRNA (regulatory T cell, T reg) and programmed cell death ligand-1 (PD-L1) mRNA were significantly increased in the HCV-high group. HCV-high livers had two molecular immune responses: increased APC numbers and adaptive immunity and the induction of immune tolerance. The local hepatic imbalance of contradictory immune responses might be responsible for high HCV loads.

To establish a diagnostic index for predicting enzootic bovine leukosis (EBL), proviral bovine leukemia virus (BLV) copies in whole blood, lymph nodes and spleen were examined by quantitative real-time PCR (qPCR). Cattle were divided into two groups, EBL and BLV-infected, based on meat inspection data. The number of BLV copies in all specimens of EBL cattle was significantly higher than those of BLV-infected cattle (p < 0.0001), and the number of BLV copies in the lymph nodes was particularly large. Over 70 % of the superficial cervical, medial iliac and jejunal lymph nodes from EBL cattle had more than 1,000 copies/10 ng DNA, whereas lymph nodes from BLV-infected cattle did not. These findings suggest that the cattle harboring more than 1,000 BLV copies may be diagnosed with EBL.