Analyses of viral inter- and intra-host mutations could better guide the prevention and control of infectious diseases. For a long time, studies on viral evolution have focused on viral inter-host variations. Next-generation sequencing has accelerated the investigations of viral intra-host diversity. However, the theoretical basis and dynamic characteristics of viral intra-host mutations remain unknown. Here, using serial passages of the SA14-14-2 vaccine strain of Japanese encephalitis virus (JEV) as the in vitro model, the distribution characteristics of 1,788 detected intra-host single-nucleotide variations (iSNVs) and their mutated frequencies from 477 deep-sequenced samples were analyzed. Our results revealed that in adaptive (baby hamster kidney (BHK)) cells, JEV is under a nearly neutral selection pressure, and both non-synonymous and synonymous mutations represent an S-shaped growth trend over time. A higher positive selection pressure was observed in the nonadaptive (C6/36) cells, and logarithmic growth in non-synonymous iSNVs and linear growth in synonymous iSNVs were observed over time. Moreover, the mutation rates of the NS4B protein and the untranslated region (UTR) of the JEV are significantly different between BHK and C6/36 cells, suggesting that viral selection pressure is regulated by different cellular environments. In addition, no significant difference was detected in the distribution of mutated frequencies of iSNVs between BHK and C6/36 cells.

Human astroviruses (HAstVs) were first identified in 1975 and can be classified into three clades: classic HAstVs (HAstV 1-8), MLB (MLB1-3) and VA (VA1-5), with MLB and VA were newly identified. Recombination and a high mutation rate make HAstV as one of the rapidly evolving infectious agents. This study reported a novel identified recombinant human astrovirus (Y/1-CHN) and its long existence in two immunocompromised patients with diarrhea following allogeneic hematopoietic stem cell transplantation (allo-HSCT). The identified Yu/1-CHN genome contains 6801 base pairs encoding three open reading frames, with ORF1a best hit to the HAstV1 (Pune strain, 97 % nucleotide identity), while ORF1b and ORF2 best hit to HAstV-5 (DL30 strain, 99 % nucleotide identity). Possible recombination breakpoint was predicted to be located in the boundary of ORF1a and ORF1b. Different quasispecies were found in the host, and the dN/dS ratios of the S and P domains were determined to be 1.189 and 1.444, respectively, suggesting a positive selection existed. Fecal samples collected in different clinical phases from the two patients were all positive for Yu/1-CHN, suggesting a long existence of the virus in the host. It was indicated that immunocompromised patients may a reservoir for astrovirus, their excreta should be monitored even after discharge from hospital.

Peste des petits ruminants (PPR) is an highly contagious disease of small ruminants, and caused by peste des petits ruminants virus (PPRV), a member of the genus Morbillivirus in the family Paramyxoviridae. The first outbreak of PPR in China was officially reported in July 2007, when a PPRV strain was successfully isolated from a sick goat in Tibet, followed by sequencing at a full-genome level (China/Tibet/Geg/07-30, GenBank: FJ905304.1). To date, this isolate has been virulently attenuated by more than 90 serial passages in Vero-Dog-SLAM cells at our laboratory. In this study, a total of nine strains by serial passages (namely the 10th, 20th, 30th, 40th, 50th, 60th, 70th, 80th, and 90th passages) were chosen for sequencing of six structural genes in PPRV. The sequence analysis showed that mutation rates in all viral genes were relatively low, and only a few identical mutations within certain genes were stably maintained after an earlier passage, perhaps indicating a predominance of mutants after such a passage.

Genetic variation in both structural and nonstructural genes is a key factor in the capacity of porcine reproductive and respiratory syndrome virus (PRRSV) to evade host defenses and maintain within animals, farms and metapopulations. However, the exact mechanisms by which genetic variation contribute to immune evasion remain unclear. In a study to understand the role of host genetics in disease resistance, a population of pigs were experimentally infected with a type 2 PRRSV isolate. Four pigs that showed virus rebound at 42days post-infection (dpi) were analyzed by 454 sequencing to characterize the rebound quasispecies. Deep sequencing of variable regions in nsp1, nsp2, ORF3 and ORF5 showed the largest number of nucleotide substitutions at day 28 compared to days 4 and 42 post-infection. Differences were also found in genetic variations when comparing tonsil versus serum. The results of dN/dS ratios showed that the same regions evolved under negative selection. However, eight amino acid sites were identified as possessing significant levels of positive selection, including A27V and N32S substitutions in the GP5 ectodomain region. These changes may alter GP5 peptide signal sequence processing and N-glycosylation, respectively. The results indicate that the greatest genetic diversity occurs during the transition between acute and rebound stages of infection, and the introduction of mutations that may result in a gain of fitness provides a potential mechanism for persistence.