*2.1. Clinical Manifestation*

Pigs in Group 1 (co-infected\_P1) were inoculated with equal amounts of the TD/96/TWN strain (designated TD/96 and belonging to genotype 2.1) and the 94.4/IL/94/TWN strain (designated 94.4 and belonging to genotype 3.4) simultaneously. Pigs in Group 2 (co-infected\_P2) were inoculated with whole blood taken from a pig of Group 1 at 12 dpi. Pigs in Group 3 (co-infected with Ab) were inoculated with equal amounts of the TD/96 strain and the 94.4 strain simultaneously, born from a sow vaccinated with LPC vaccine, in which the maternal antibody response was in decline.

Clinical scores and temperature records of the experimental pigs are shown in Figure 1 and Table 1. The clinical signs were most numerous and significantly severe in co-infected\_P2 (second passage of competition) pigs (Group 2; average maximum clinical score: 19.33 ± 0.58) and the first febrile reaction was detected as early as 2 or 3 days post-infection (dpi). The highest clinical score of Group 2 was the result of the pigs being inoculated with higher viral loads of the TD/96 strain than the pigs of the other two groups were (Table 1).

**Figure 1.** Body temperatures of pigs co-inoculated with classical swine fever viruses of two genotypes. Pigs in the group co-infected\_P1 (square) were inoculated with TD/96 strain (genotype 2.1) and 94.4 strain (genotype 3.4). Pigs in the group co-infected\_P2 (triangle) were inoculated with whole blood taken from a pig of group co-infected\_P1 at 12 days post-infection. Pigs in the group co-infected with Ab (circle) were born from a sow vaccinated with LPC vaccine and were inoculated with the two virus strains. Each point represents the mean and standard deviation of the three pigs in the same group.

Those pigs in co-infected\_P1 (first passage of competition) were less severe (Group 1; average maximum clinical score: 15.67 ± 1.53), and first febrile reaction was detected at 4 to 6 dpi. Clinical signs of those pigs co-infected with the presence of maternal antibodies developed more slowly and were significantly less severe (Group 3; average maximum clinical score: 4.67 ± 0.58) and all pigs survived until the end of experiment. First febrile reaction in pigs in Group 3 was detected at 6 to 12 dpi.

The febrile profile shown in Figure 1 was largely compatible with those of the averaged clinical scores shown in Table 1. In other words, Group 2, having higher clinical scores, also had higher febrile reactions, while Group 3, having lower clinical scores, also had lower febrile reaction.

## *2.2. Virus Titration of Viremia for Co-Infected\_P2 Pigs Inoculation*

Co-infected\_P2 pigs were inoculated with whole blood taken from a pig of the co-infected\_P1 group at 12 dpi (Group 2, Table 1). The virus titers of the TD/96 strain and the 94.4 strain from viremia at 12 dpi of a co-infected\_P1 pig were 108.3 TCID50/mL (tissue culture infectious dose 50%) and 105.87 TCID50/mL, respectively, determined by CSFV genotype-specific monoclonal antibodies (mAbs) (Table 2).



indicate a statistically significant difference (*<sup>p</sup>* < 0.05) from each other. The superscript letter "a" indicates the highest viral load and "c" indicates the lowest viral load among compared groups. ¶ The pig was euthanized at the end of the experiment. § ND: not detected.

#### *Pathogens* **2020**, *9*, 261



† TD/96 and 94.4 indicate the TD/96/TWN and 94.4/IL/94/TWN strains, respectively. \* IFA indicates indirect fluorescent assay. ¶ RT-MRT-PCR indicates reverse transcription multiplex real-time polymerase chain reaction.

## *2.3. Cross-Neutralizing Antibodies against Three Genotypes of CSFVs*

The role of maternal antibodies during dual infections was further investigated (Table 3). The pigs' sera of co-infected\_P1 (Group 1) and co-infected\_P2 (Group 2) at 0 dpi to the end of the experimental period showed no neutralizing antibodies against genotypes 1.1 (LPC/AHRI strain), 2.1 (TD/96 strain) or 3.4 (94.4 strain), consistent with the more severe clinical scores and animal mortalities at 9–13 dpi observed in both groups (Table 1).

**Table 3.** Cross-neutralizing antibodies of three pigs co-inoculated with classical swine fever viruses with maternally derived neutralizing antibodies (Group 3).


† LPC, TD/96 and 94.4 indicate the LPC/AHRI, TD/96/TWN and 94.4/IL/94/TWN strains, respectively. \* Values with different superscript letters, a–b, among the three groups of samples at the same dpi indicate a statistically significant difference (*p* < 0.05) from each other. The superscript letter "a" indicates the highest viral load, and "b" indicates the lowest viral load among the compared groups, while "ab" indicates a viral load in between categories "a" and "b". No significant difference exists between values containing the same letter. The absence of a superscript letter indicates no statistical analysis.

The neutralizing antibodies against the LPC strain in the sera of co-infected pigs with maternal antibodies (Group 3) before inoculation indicated that these pigs were in a declining phase of maternal antibody response (data not shown). Sera of co-infected pigs at 0–6 dpi did show cross-neutralizing antibodies against three genotypes (Table 3), consistent with the much milder clinical scores seen in this group. A critical time window was noted at 6–8 dpi, when the cross-neutralizing antibody titers against TD/96 and 94.4 CSFVs dropped from log2 2.3–2.8 to < 2.0 (Table 3), and yet the animals survived until 14 dpi (Table 1). The neutralizing antibody titers against the LPC strain were significantly higher than those against the TD/96 strain and the 94.4 strain at 0 and 2 dpi and significantly higher than those against the 94.4 strain at 4 and 6 dpi. There was no significant difference between the neutralizing antibody titer against the TD/96 strain and the 94.4 strain. From 8 or 10 dpi to 14 dpi, sera of co-infected with Ab pigs did not show detectable neutralizing antibodies against any of the three genotypes (Table 3).
